Russia said it was a retaliatory move after hostility from the Czech Republic
Russia expelled two Czech diplomats on Monday in a tit-for-tat move triggered by a fake story about a poisoning plot.
The Russian Foreign Ministry said in a statement that it had summoned the Czech ambassador to the country to inform him of the move.
The two Czech diplomats, now persona non grata, and their families have until Wednesday to leave the country.
This is "retaliatory measures in connection with Prague's hostiles actions" on the basis "of absurd and unsubstantiated charges", the ministry said.
The Czech government ordered the expulsion of two Russian embassy employees on June 5 over allegations of spreading false information about a plot to murder three Prague politicians, including the city's mayor Zdenek Hrib.
The false rumour was also reported on the Czech media, which claimed Russia got "irritated" over the decision to remove the statue of a Soviet commander who helped to liberate Czech Republic from the Nazis, Ivan Stepanovic Konev.
As a result, Prague's mayor and two other local politicians, Pavel Novotny Ondrej Kolar, were placed under police protection.
Despite the false murder plot rumours, the removal of the statue sparked a diplomatic row between Russia and Czech republic, with Moscow opening an investigation for "public desecration of the symbols of Russian military glory".
Russian Defense Minister Sergei Shoigu demanded that Prague hand the statue over to Moscow.
Another controversial episode took place in February, when a Prague square in front of the Russian Embassy was renamed after slain Russian opposition leader Boris Nemtsov, who got killed in 2015.