MSF is the latest in a string of Western-funded charities, rights groups and non-government organisations (NGOs) to halt operations in Russia since Moscow's invasion of Ukraine in February 2022.
Last year, Russia declared NGOs Greenpeace, Transparency International and WWF "undesirable", effectively banning them from operating in the country.
"It is with a heavy heart that we have to close our activities in Russia," MSF said late on Monday, adding that it had overseen dozens of programmes there since 1992.
The charity said it had received a letter last month from the justice ministry removing it from the register of foreign NGOs in Russia.
The decision applied to the Dutch branch of MSF in Russia but since this was the only association active in the country, it effectively ended the group's presence there.
There is still a "branch office" dealing with non-operational activities in Russia, MSF spokeswoman Maria Borscheva told AFP.
"To comply with the decision, we had to close our operations in the country and terminate the contracts of our staff in accordance with all legal requirements," she said.
Since the conflict with Ukraine erupted in 2022, MSF said it had provided humanitarian aid to more than 52,000 refugees and displaced people.
More than 15,400 people had received free medical, mental health and psychosocial support, the group said.
Borscheva said more than 50 staff were working in Russia. According to the MSF website, it spent 3.4 million euros ($3.8 million) on programmes in the country last year.
"We are very sad to conclude our programmes in the country as many people in need of medical and humanitarian assistance will now be left without the support we could have provided to them," said Norman Sitali, MSF operations manager in Russia.
"MSF would like to still work in Russia again, if and when possible," added Sitali.
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