Almost 300 lorries of humanitarian aid have left the Moscow area bound for eastern Ukraine, Russia has said.
Russian media said the cargo, including hundreds of tonnes of grain, baby food, power generators and medicine, will go to civilians trapped by fighting in the area held by pro-Russia rebels.
Russian officials said the Ukrainian government had agreed to the operation.
Western powers have warned Russia not to use humanitarian assistance as a pretext to invade eastern Ukraine.
Russian President Vladimir Putin said on Monday that Russia was launching a humanitarian mission in co-operation with the International Red Cross.
The Red Cross had acknowledged the situation in eastern Ukraine was critical with thousands of people reported to be without access to water, electricity and medical aid.
It said it had agreed in principle to such an operation, but made it clear the practical details needed to be clarified first.
Media reports said the cargo left the town of Naro-Fominsk, south-west of Moscow, on Tuesday morning.
"The convoy will deliver to the residents of eastern Ukraine about 2,000 tonnes of humanitarian cargo, collected by the residents of Moscow city and region," Moscow region officials said.
At least 1,500 people have died since Ukraine's new government sent in troops to put down an insurrection by pro-Russia separatists in the Donetsk and Luhansk regions in mid-April.
Pro-Russia separatists sparked a four-month battle with Ukrainian government forces when they overran large swathes of the east and declared independence from Ukraine.
The fighting has displaced hundreds of thousands of people, many of whom have fled to Russia.
Ukrainian forces have now encircled Donetsk, a city of one million people before the unrest began, and residents are struggling without power or reliable sources of food.
The fighting has prevented a full investigation of the MH17 air disaster, in which 298 people died.
The Malaysia Airlines plane was brought down in the conflict zone in eastern Ukraine on 17 July and the remains of many of the victims are still to be recovered.
It is strongly suspected that the plane was shot down by pro-Russian rebels. Russia and the separatists have blamed the Ukrainian military.
Speaking on Tuesday, US Secretary of State John Kerry said he hoped to find a way for Ukraine to work with Russia to help bring about a formal investigation into the crash.
(BBC)