Gloucester have had two points deducted from their total for the 2014-15 Aviva Premiership season for fielding an ineligible player upon a successful appeal by the Rugby Football Union.
The club appeared before an independent panel in April after erroneously selecting lock Mariano Galarza for their 23-6 defeat at Sale on March 29 and subsequently received a £5,000 fine and a deduction of two league points suspended until the end of 2016-17 campaign.
However, the hearing also decided the points deduction would only be activated if Gloucester repeated the offence, resulting in an appeal from the RFU on the grounds that the circumstances were not so exceptional to justify the suspension.
An independent appeal panel agreed with the RFU, ordering there should not be any suspension of the points deduction.
"This part of the appeal was unopposed by Gloucester but the cross-appeal by the club, that it should only have a single point deducted, was dismissed," an RFU statement read.
"Accordingly, Gloucester will be deducted two league points for this season (2014-2015) immediately. The £5,000 fine also remains payable, plus the first-instance hearing costs of £500."
The removal of two points has no impact on Gloucester's final position of ninth with 10th-placed London Irish finishing eight points behind before the appeal's decision.
Gloucester have responded to the success of the RFU's appeal by expressing their dismay at the outcome.
"We're naturally disappointed by the RFU's decision to appeal the original outcome and the subsequent decision," chief executive Stephen Vaughan said.
"There is no point revisiting the reasons why we feel this is unduly harsh, but can only hope that the reasons were fact based and not due to pressure from other parties.
"In the interests of moving on, and looking forward to the start of a new season, we have accepted the decision and now consider the matter closed."