The school was founded in 1565 by a Royal Charter of Elizabeth I whose letters patent, sealed on 29 January, authorised Sir Roger Cholmeley to establish the ‘’’Free Grammar School of Sir Roger Cholmeley, Knight at Highgate’’’.
Cholmeley, a former Chief Justice and local landowner, decided to found a charitable school “for the good education and instruction of boys and young men” in Highgate and the local parishes. On 27 April 1565 he was granted by Edmund Grindal, the Bishop of London, some land on the site of the old gatehouse to the Bishop's park and hermit's chapel (opposite the Gatehouse Inn, which still exists). A new chapel and buildings for the school and the local curate, who was expected to be the teacher, were built. The chapel also served as a chapel of ease for Highgate residents.
However, by the early nineteenth century a dispute arose because the charity was spending more money, and the curate more time, on the local chapel than on the pupils. A House of Commons commission visited in 1819 and found the Master, the Rev Samuel Mence, was paying a sexton to teach the boys. In a long and bitter action brought in the High Court against the Trustees it was contended that this was contrary to its founding charitable deed. Lord Chancellor Eldon, in his 1827 judgment, agreed, finding "the charity is for the sustenance and maintenance of a free Grammar school". The trustees were forced to comply and a separate local church for Highgate, St Michael's, was built in South Grove after a successful local appeal. Mence struggled on at the school until 1838 when there were only 19 pupils.
An expansion of the school occurred under the next headmaster Rev Dr John Bradley Dyne (Fellow of Wadham College, Oxford) between 1838–1874. Under Dyne, by the 1870s the school had largely dropped free provision for local parish boys and alongside the day places boarding was encouraged for boys from the upper and upper middle classes; fees were introduced and academic standards improved.In the period up to this time the school was known commonly as the Free Grammar School at Highgate, the Highgate Grammar School, or the Cholmeley School. Like other public schools, Highgate followed Dr Arnold at Rugby School in introducing the house system. Also like other public schools, Dyne mercilessly flogged the pupils with a birch rod.
In the 1860s land was acquired in Bishopswood Road, which provided extensive sports fields and on which several boarding houses and private residences were built. During this period the current chapel and main buildings were erected, designed by Reginald Blomfield (who had also designed Lady Margaret Hall, Oxford). A fragment of the older school building, a gateway with a rusted bell mechanism above between the porter's lodge and the main school building, remained intact until 2006 when the bell was refurbished and the old entrance itself rebuilt in a more modern style. The senior school continues to occupy today the island site in Highgate Village on which it was founded.
During the Second World War the school's buildings were commandeered by the British government and the school was evacuated to Westward Ho! in Devon, returning to Highgate in 1943. This return was maybe slightly premature because one afternoon in 1944 a V‑1 Doodlebug flying bomb landed and exploded in the field behind the Junior School. Luckily, the only serious casualty was a cricket scorebox.
The poet Samuel Taylor Coleridge was buried in the school chapel, his grandson an Old Cholmeleian. However, in 1965 after a row with the council there was a ceremonial disinterring of Coleridge at which the then Poet Laureate John Masefield spoke and the remains were reburied at St Michael's parish church just a few hundred yards away. There are currently weekly services for pupils and staff in the Chapel, and monthly celebrations of Evensong, which are open to the general public. There are also twice-termly Communion services.
Highgate School has the oldest Public School freemasons' lodge, Cholmeley Lodge No 1731, formed in 1878, part of the Public Schools Lodges Council.
Until recently the school had two blocks of Eton Fives courts, one structure with ten courts (of which six were built in 1899 and a further four added c.1913); a second block of eight courts constructed in the 1920s was removed in 2014.
Boarding and weekly boarding at Highgate declined in the years up to the early 1990s when the last boarders left. In 1993 one of the former houses was converted to create the coeducational pre-preparatory school.
In 2001 the school announced its intention to become fully co-educational ending over four hundred years of single sex education, and girls joined the Senior and Junior schools from 2004. According to the Good Schools Guide "Its decision to go co-ed has helped to put its popularity and academic standards on upward trajectories".
In April 2006 the Mills Centre for Art, Design and Technology was opened, incorporating an area commemorating former director of art Sir Kyffin Williams.
In January 2013 the Charter building was opened by former pupil and Governor Lord Hill.
In May 2014 the Sir Martin Gilbert Library was opened by former Prime Minister Gordon Brown.
Throughout 2015 the school celebrated its 450th anniversary. In January 2015 a museum opened, which can be visited by the public on Saturday mornings in term-time.
In September 2016 a new building for the Junior School opened.