Italian Prime Minister Mario Draghi said that "war crimes" carried out in Ukraine must be punished, while Rome said it was expelling 30 Russian diplomats on national security grounds.
In a speech in Turin on Tuesday, Draghi also urged Russian President Vladimir Putin to halt the "massacre of civilians".
"President Putin, the Russian authorities and its army will have to be held to account for their actions," he said.
Draghi called for an independent investigation into deaths in the Ukrainian town of Bucha which came to light on Sunday, prompting international outrage.
Russia has denied responsibility for the Bucha killings.
Draghi said Italy was aligned with the European Union in its stance over the hostilities in Ukraine and "supports with conviction" a new package of sanctions presented by the EU Commission earlier on Tuesday.
Italy said earlier that it was the latest Western country to expel Russian diplomats because of security concerns following the invasion of Ukraine in February.
The Russian embassy in Rome said that no proof had been provided to back up the security concerns and that diplomats and their families had been given only 72 hours to leave Italian territory.
Ambassador Sergey Razov warned that the move would further harm bilateral relations and that Russia would respond.
(Reporting by Angelo Amante; Writing by Keith Weir; Editing by Agnieszka Flak and Emelia Sithole-Matarise)
