Luca Attanasio, 43, died in hospital on Monday after the United Nations convoy he was travelling in came under fire near Goma, a statement said.
The convoy reportedly belonged to the UN's World Food Programme (WFP).
An Italian military police officer travelling with the envoy and a Congolese driver were also killed.
"It is with deep sadness that the foreign ministry confirms the death, today in Goma, of the Italian ambassador," the ministry's statement said.
The attack in North Kivu province is believed to have been an attempted kidnapping, according to officials at the nearby Virunga National Park.
Mr Attanasio was a passenger in one of two vehicles travelling about 15km (nine miles) north of Goma when the attack happened on Monday morning.
The vehicles were "ambushed by a group of six attackers, who fired warning shots before taking all the passengers into the Virunga National Park", the governor of North Kivu province, Carly Nzanzu, told the BBC.
"That's when a patrol of Virunga National Park rangers intervened and managed to free four people," he said.
"Unfortunately, before they ran away, the assailants shot the ambassador and his bodyguard, and they killed the Congolese driver at the start of the attack," the governor added.
The WFP said Mr Attanasio was travelling from Goma to visit a "schools programme" in the village of Rutshuru in eastern DR Congo.
"The attack... occurred on a road that had previously been cleared for travel without security escorts," the Rome-based agency said in a statement.
The two other fatalities were military police officer Vittorio Iacovacci, 30, who had been serving at the embassy since last September, and their Congolese driver, whose name has not yet been officially released.
A number of other passengers were reportedly injured.
It is not clear who was behind the attack, but the interior ministry has accused a Hutu rebel group known as the Democratic Forces for the Liberation of Rwanda (FDLR).
Many armed groups are known to operate in and around the park and militias clash regularly in the east of the country, where a large UN force is struggling to keep the peace.
The DRC army has deployed troops to help search the area.