Bristol Bridge is now closed to general traffic - with motorists caught using the route risking a £60 fine.
The closure by Bristol City Council is aimed at easing congestion and reducing pollution in the city centre.
Taxis and buses will continue to be able to use the route, on Victoria Street.
But motorists who flout the new rule will face penalties with the council planning to bring in cameras at the end of Baldwin Street and in Union Street.
Those caught will be fined £60, but will be given an option to pay half if paid up within 14 days.
The 'initial weeks' will only see warning letters sent out to drivers to allow people to get used to the closure.
The closure has been brought in alongside the pedestrianisation of the Old City and are part of a package of major changes being accelerated to transform Bristol’s transport network.
Councillor Kye Dudd, cabinet member for transport, said: “All of these major changes to key Bristol roads will make a massive difference to the quality of our air as we divert traffic away from the city centre.
“These transformative measures will create a more connected future for everyone, with cleaner air, safer and better public transport, and improved walking and cycling routes. They will also support social distancing during the Covid-19 pandemic.
“These important changes will inevitably result in a period of adjustment as we all adapt, but they are crucial for protecting public health, encouraging more efficient travel, and making bus services more reliable in the long-term.
James Freeman, managing director at First West of England, said: “At a time when it’s possible for everybody to return to the buses again after the lockdown, it is great to see Bristol City Council extending bus priority in this way.
“We are taking immediate advantage of this by doubling the number of route 2 buses that cross Bristol Bridge from today.
"We’re doing this by introducing a new 2a route from Stockwood to Southmead, which runs parallels to the number 2 for most of its route.”
The council has said the measures are being done on a temporary basis initially and it will assess how they are working before permanent changes are made.
