From 0300 GMT Saturday, cars, lorries and other private vehicles were barred from using the last trunk of the avenue Via dei Fori Imperiali, which links Piazza Venezia to the Roman amphitheatre.
Traffic has been diverted to an adjacent route and only public transport will be allowed on the old route.
The decision was taken by the new mayor of Rome, Ignazio Marino of the leftist Democratic Party, who would like eventually to make the Via dei Fori Imperiali a pedestrian area.
The number of visitors to the Colosseum, the biggest ancient Roman amphitheatre ever built, has increased from a million to around six million a year over the past decade, thanks mainly to the 2000 blockbuster film "Gladiator".
But it has also fallen into disrepair in recent years: bits of stone, blackened by pollution, have fallen off and some experts have voiced concern that the foundations are sinking, and the amphitheatre is starting to lean.
Long-delayed repairs to the 2,000-year-old monument, funded by Italian billionaire Diego Della Valle, are in the pipeline — but problems with red tape mean they have yet to get off the ground.