Neill Collins's side have been knocked out of the FA Cup for fielding an ineligible player against non-league side Horsham in a first-round replay victory.
Following the Tykes' removal, Horsham will proceed to play Sutton United in round two of the FA Cup, subject to any appeal from Barnsley.
The player in question was not correctly registered by the club for the match, which is in direct violation of FA Cup Rule 103.
A statement from Barnsley read: "We would like to apologise to our fans, players and staff for this regrettable error. This mistake - although unintentional - falls way below the high standards we set at this club and is simply not what you deserve."
"We have already conducted a full internal investigation and have subsequently put safeguards in place to ensure this will never happen again."
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