Gloucestershire County Council says a temporary one-way system will be introduced on London Road from 10 June.
The restriction is needed to allow the next phase of the Gloucester City Centre Cycle Spine project to be carried out safely.
Westbound traffic heading towards Black Dog Way, Gloucester Transport Hub and Gloucester railway station will not be permitted, while eastbound traffic towards Gloucester Royal Hospital and the University of Gloucestershire’s Oxstalls campus will continue as normal.
A signed diversion route will be in place, with the temporary arrangement expected to remain until early October 2026.
Cyclists travelling towards the city centre will be required to dismount and use the footway during the works.
Councillor Roger Whyborn said the scheme would make cycling safer and easier, helping to reduce congestion, improve air quality and encourage more sustainable travel across Gloucestershire.