Nettles was born in St Austell, Cornwall in 1943. His birth mother was an Irish nurse who came to work in the United Kingdom during the Second World War. He was adopted at birth by carpenter Eric Nettles and his wife Elsie. As a youth he attended St Austell Grammar School. In 1962, he studied history and philosophy at the University of Southampton. There he first performed as an actor, and after graduation he joined the Royal Court Theatre.

In 1966 Nettles married his first wife, Joyce, and their daughter Emma was born in 1970. After they divorced in 1979, Joyce Nettles later became a casting Director on 23 episodes of Midsomer Murders. He married his second wife, Cathryn Sealey, in July 1995 in Evesham, Worcestershire.

Nettles played Laertes to Tom Courtenay's Hamlet in 1969 at the University Theatre for 69 Theatre Company in Manchester. From 1969 to 1970, he was in repertory at the Northcott Theatre in Exeter, and in the latter year had his first screen role in the film One More Time. The following year he played Dr. Ian Mackenzie in the period drama A Family at War, a role he continued until 1972. Following that he had small parts in many TV programmes including The Liver Birds, Dickens of London, Robin of Sherwood and an episode of Enemy at the Door called "Officers of the Law", first broadcast in March 1978. The latter was set in Guernsey during the Second World War German occupation and Nettles played a police detective ordered to work for the Germans and anguished over the conflict between his duty and collaborating with the enemy. He played fraudster Giles Sutton in ITV's Heartbeat.

In 1981, Nettles won the role that made him a household name in the UK, that of Jim Bergerac in the Jersey-set crime drama Bergerac. This ran for 87 episodes until 1991. Following the end of Bergerac Nettles did five seasons with the Royal Shakespeare Company, appearing in The Winter's Tale, The Merry Wives of Windsor, Julius Caesar, Richard III and The Devil is an Ass. In 1992, he appeared in an episode of Boon, and in 1993 appeared as Jim Bergerac in the spoof police comedy The Detectives.

During the filming of Bergerac, filmed on the island of Jersey, he wrote Bergerac's Jersey (BBC Books, 1988; ISBN 0-563-20703-5), a travel guide to filming locations in the series. He followed up with John Nettles' Jersey: A Personal View of the People and Places (BBC Books, 1992; ISBN 0-563-36318-5) about the island's landscape, personalities and history.

In 1991 he wrote the semi-autobiographical Nudity in a Public Place: Confessions of a Mini Celebrity (Robson Books; ISBN 0-7451-1961-1) about becoming a "reluctant heartthrob" to female viewers of Bergerac. This was re-released as a Kindle version on Amazon in 2014 following the reruns of Bergerac on BBC2 as part of their afternoon nostalgia collection.

In 1995, Nettles was approached by Brian True-May to play Tom Barnaby in a new murder mystery series he was to produce called Midsomer Murders. This was to be the second major role of his career, again playing a policeman. Midsomer Murders made him a household name across the world. In 2003, he played Barnaby in the Boxing Day episode of French & Saunders. Nettles appeared in a 2001 episode of Heartbeat. In 2007, he appeared in the BBC Radio 4 comedy series Will Smith Presents the Tao of Bergerac alongside Comedian Will Smith about an obsessive fan of the series.

Nettles narrated the BBC documentary series Airport from 1996 to 2005.

In early 2010, Nettles wrote, presented and produced a three-part documentary, Channel Islands at War, to mark the 70th anniversary of the German invasion and subsequent occupation of the Channel Islands. He received threatening letters from some residents of Jersey, accusing him of implying that islanders were collaborators. He defended the documentary saying "There is no possible way you could have avoided collaboration with the occupying power who had power over the civilian population. If you had not toed the line you would have been shot." This view was supported by local historians and members of the Channel Islands Occupation Society.

In February 2009, it was announced that Nettles had decided to leave Midsomer Murders after two further series were made. His final appearance on-screen was on 2 February 2011, by which time he had appeared in 81 episodes. About his departure, he commented, "It’s always wise to leave people wanting more, rather than be booed off the stage because you bored them."

Nettles was appointed Officer of the Order of the British Empire (OBE) in the 2010 Birthday Honours. On 21 September 2012 he was awarded an honorary doctorate by the University of Plymouth. He also agreed to be patron of Devon charity The Mare and Foal Sanctuary in July 2014.

In 2016 it was also announced that he would play the recurring role of Ray Penvenen in the second series of Poldark.