Mishustin confirmed his test results in a videoconference with President Vladimir Putin, and nominated his deputy to take over for him as acting Prime Minister.
"It just became known that the coronavirus tests I took came back positive, so I must oblige by the self-isolation rules and it's mandatory [that I do that] for the safety of my colleagues," Mishustin said in a segment aired on Russia 24 state channel.
Putin signed a decree appointing Andrey Belousov to the role on an acting basis, following Mishustin's recommendation.
Mishustin's diagnosis makes him the most high-profile political figure in Russia to contract the coronavirus.
The Prime Minister attended a meeting of the government's coordinating council for combating the spread of the virus on Wednesday, in which he said it was "impossible" to give an exact date when Russia's borders would re-open.
The country surpassed 100,000 confirmed cases earlier on Thursday, after announcing its highest single-day rise of 7,099 new infections.
Since the beginning of the pandemic 1,073 people have died in Russia, according to the country's coronavirus response headquarters.