From now on, club officials face a one-year suspensionn for ignoring the rule which obliges teams to field at least one under-21 player.
A second offence would result in a life ban, the RFU said
"We had to act immediately so no one would try to circumvent the rule on purpose," said RFU chief Sergei Fursenko.
Zenit fell foul in Sunday's 1-1 draw at home to CSKA Moscow and on Wednesday the RFU's disciplinary committee handed them a 3-0 defeat and fined them 200,000 roubles ($7,100).
Zenit coach Luciano Spalletti said he had ordered his staff to ignore the rule because he felt the club's under-21 players were not good enough to face CSKA.
The flamboyant Italian said he was willing to pay the fine himself but said he was unaware it would also cost his team the points.
Sunday's defeat dropped Zenit from second to fifth place, three points behind surprise league leaders Volga Nizhny Novgorod.
On Monday, the Russian champions fired former international Vladislav Radimov, who as the club's administrator was responsible for filling in the names in the team's line-up.
Spalletti, who last year led Zenit to a domestic league and cup double in his first season, has been heavily criticised in the Russian media and RFU's ethics committee chairman Alu Alkhanov has called for an investigation.
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