"Stop the massacre, stop complicity!" read a banner held by protesters at the start of the march, amid a sea of red, white and green Palestinian flags, peace flags and "Free Palestine" signs.
The peaceful protest attracted a crowd estimated by organisers at 300,000 people.
Police later said those estimates were "largely confirmed", according to Italian news agency AGI.
The crowd made its way from Rome's central Piazza Vittorio to San Giovanni, where speakers took to a stage to call for an end to violence and denounce the "silence" of Prime Minister Giorgia Meloni's government.
The leader of the centre-left Democratic Party, Elly Schlein, called the turnout "an enormous popular response" in opposition to the war.
Italy's Five Star Movement and the Greens–Left Alliance were also behind the protest.
The demonstration aimed to "say enough to the massacre of Palestinians [and] to the crimes of Netanyahu's far-right government," Schlein told journalists.
It was also intended to show the world "another Italy," one that "wants peace, an immediate ceasefire, the release of all hostages, humanitarian aid, and recognition of the state of Palestine," she said.
The leader of the Five Star Movement, Giuseppe Conte, told the crowd he was there "to not be an accomplice in genocide".
Israel has come under increasing international criticism over the dire humanitarian situation in the Palestinian territory, where the United Nations warned in May that the entire population was at risk of famine.
In Italy, Meloni has been pressured by the opposition into condemning the actions of Israel's leader Benjamin Netanyahu in Gaza – but her criticism has been tempered.
Last month, Meloni called the humanitarian situation in Gaza "increasingly dramatic and unjustifiable", saying she'd had "difficult conversations" with Netanyahu, while at the same time noting that "it was not Israel that started the hostilities".
Many protesters came from across Italy to participate in the demonstration in the capital, including Gabriella Branca, a lawyer from Genoa.
"It's unbearable to witness the massacre of 60,000 people, including 20,000 children. We have to say enough," the 67-year-old told AFP.
"In other countries demonstrations have drawn millions of people, so I hope that today in Rome we can send a signal to all of Italy, so that everyone takes to the streets to say enough and, above all, to try to find peace," she said.
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