Although both sides have reported cross-border shootings in the past, the incident appears to be the first time Moscow has reported fatalities on its side of the border from the three-month conflict which has killed hundreds of people in Ukraine.
Ukraine denied its forces had fired across the border and suggested such an attack could have been the work of rebels trying to provoke Moscow into intervening on their behalf. The rebels denied they were responsible.
The bellicose rhetorical response from Moscow raises the renewed prospect of overt Russian intervention, after weeks in which president Vladimir Putin had appeared intent on disengaging, pulling back tens of thousands of troops he had massed at the frontier.
Russia sent Ukraine a note of protest describing the incident as "an aggressive act by the Ukrainian side against sovereign Russian territory and the citizens of the Russian Federation," the Russian Foreign Ministry said in a statement warning of "irreversible consequences".
Russian deputy foreign minister Grigory Karasin added: "This represents a qualitative escalation of the danger to our citizens, now even on our own territory. Of course this naturally cannot pass without a response.
"What has taken place once again reinforces the need for an urgent end to the bloodshed." He called for talks between "the sides in the conflict", language Moscow uses to demand Kiev recognise the rebel leaders - many of them Russians who crossed the border to fight - as legitimate interlocutors.
Russia's Investigative Committee, a crime-fighting body answering directly to Mr Putin, said it had launched a criminal case for murder after a shell landed in the yard of a house on the Russian side of the frontier.