"We have chosen to expel four diplomats, four particular diplomats, in order to send a clear and proportionate signal to the Russian government about the seriousness of this case," he said, adding that he was grateful for strong EU support.
"We will discuss with partners the need for future EU-Russia engagement to take our concerns on this case into account."
Aside from the expulsions - the first since 1996 – Britain said it would make it harder for Russian officials to enter the country and review co-operation on other issues.
Gordon Brown, the prime minister, speaking after meeting the German chancellor, Angela Merkel, in Berlin, made no apologies for the expulsions, saying he wanted good relations with Russia, but also wanted justice to be served.
"We want to work together with Russia constructively on all the major international issues that we face," Brown said, mentioning Iran and the Middle East.
But, he added: "A murder did take place. It has been investigated by the independent prosecuting authorities. They have laid a charge and they have made it clear who they want to try for this crime.
"We're sad that the co-operation has not been forthcoming. We have therefore had to take the action that we have taken."
British prosecutors want to charge Andrei Lugovoi, a former Russian state security agent, with the murder of Litvinenko, a British citizen who died in a London hospital after ingesting a lethal dose of the rare radioactive isotope polonium-210.
Russia has rejected Britain's request to hand over Lugovoi, saying its constitution does not allow extraditions of Russian citizens.
Britain has rejected a Russian offer to put Lugovoi on trial at home, saying it doubts Moscow's promises of a fair trial.
Vladimir Putin, the Russian president, has said the demand for Lugovoi was politically motivated.
Lugovoi maintained his innocence on Monday.
Mikhail Kaminin, a spokesman for the Russian foreign ministry, said after Miliband's announcement: "They should understand well in London that the provocative actions conceived by the British authorities will not go unanswered and cannot fail to produce the most serious consequences for Russian-British relations as a whole."
He said Sergei Lavrov, the foreign minister, had relayed the same message to Miliband in a telephone call.
Interfax news agency, however, quoted an "informed source in Moscow" - a traditional reference to high-level leaks - as saying the Russian response would not necessarily be tit-for-tat.
"This is not our principle," said the source. "It would bring us back to the days of the Cold War. It's a shame that some have this principle entrenched deep in their minds."
Analysts echoed the sentiment, saying while the expulsions would provoke a robust response from Russia, they did not expect the row to escalate too far as it might harm commercial ties.