The rail crossing on Brayford Wharf East is closed to all for six weeks from today due to ongoing work on a new footbridge.
The road closed to drivers in October as work began on the multi million pound bridge.
Now, pedestrians and cyclists will also have to find another route, such as the High Street level crossing or the footbridge on the university campus.
The road was fully blocked off on Wednesday, January 2 and will remain closed until February 18.
Contractors will now begin piling, work on the barriers and traffic lights.
The project will see a stepped footbridge installed at the crossing, which will provide a new 24/7 route across the railway with improved safety.
It will not include a lift, a decision which proved controversial in the planning stages of the project.
Route Managing Director at Network Rob McIntosh said previously: “Work on this vital project, which will better connect the city of Lincoln and improve safety at the level crossing, is progressing well.
“We have worked incredibly hard to keep disruption to a minimum, however to allow the next phase of work to be carried out safely, we need to close the crossing to pedestrians and cyclists.
“We’d like to thank users for their patience whilst this work takes place and we look forward to users reaping the benefits once the project completes.”
One business on the route is taking the road closure as an opportunity to carry out refurbishment works.
The Spar shop on Brayford Wharf East will be closed until January 17.