Couples have been decorating the Ponte Milvio bridge on the river Tiber with locks for several years.
They are meant to symbolise the locking of hearts, inspired by a story described in a novel.
The city council said rust from the locks, which hang off chains, is harming the fabric of the bridge.
The custom is inspired by a book by novelist Federico Moccia in which a couple place a bicycle lock around a lamppost and throw the key into the Tiber.
The gesture was meant to symbolise the couple eternally locking their hearts together.
It took off and clusters of padlocks can be found near other landmarks in other Italian cities.
It is not the first time city authorities have stepped-in to break the locks apart.
Last year the council started a round of padlock cutting after complaints from residents that it equated to vandalism.
In 2007 the then mayor of Rome introduced a 50 euro (£40) a fine on couples found attaching padlocks to the bridge.
The BBC's Rome correspondent, Alan Johnston, says many people in Rome like the custom, and the writer behind the trend has said the locks ought to be left alone.

Spaniard Garcia, playing his first event in Europe since winning the Masters, shot a two-under 70 on Friday, leaving him eight under par overall.
Sweden's Stenson, the 2016 winner, carded a three-under 69 in his second round as he continued preparations for a defence of his Open title in July.
The pair are a shot behind Swede Joakim Lagergren and France's Joel Stalter.
England's Tommy Fleetwood, who finished fourth at the US Open on Sunday, is tied for 11th, three strokes adrift of the leaders in Munich.
But compatriot Richard Bland moved level with Garcia and Stenson with a second-round 69.
Fellow Englishman Matthew Southgate was one shot further back on seven under par overall.
"I'm not on top of my game by any means but I think it was a good kind of professional fighting display," Stenson said.
"We kept it pretty tidy, anyway, and made a couple of birdies when we had the chances and didn't drop too many. You don't have to be ashamed of three under I guess around here."
Thousands of "love padlocks" on a Roman bridge are being removed with bolt-cutters in order to protect the ancient structure.
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Sergio Garcia and Henrik Stenson are one shot off the pace after the second round of the BMW International.