Fish will be relocated during the work at Tinsley Locks using electrofishing - when a low voltage stuns them and allows them to be moved.
The 30-year-old gates on the Sheffield and Tinsley Canal will then be removed with a crane and new ones installed.
The work - on locks 5, 6, 10 and 11 - is being carried out by the Canal and River Trust.
The stretch of waterway, which opened in 1819, featured in the opening scenes of film The Full Monty in 1997.
Jon Horsfall, waterway manager for the trust, said: "This is skilled work today so it's simply incredible how the original canal builders created the canal.
"Their legacy lives on and now repairing the lock gates at Tinsley is part of our essential maintenance to enable the local canal and river network to be enjoyed by thousands of people every day."
A well-known section of canal in Sheffield is being drained ahead of a £500,000 lock repair project.