Robert Lewandowski will miss Barcelona's LaLiga trip to Atletico Madrid on Sunday after his three-game suspension was upheld by Spain's Sports Administrative Court.
The Poland striker was issued the extended ban for a gesture made towards referee Gil Manzano after being shown two yellow cards in November's 2-1 win at Osasuna.
Barcelona appealed the Spanish Football Federation's decision and Lewandowski was allowed to play in last week's 1-1 draw with Espanyol, much to the Periquitos' anger.
Head coach Xavi said on Tuesday he was hopeful the 34-year-old, who has 18 goals in 20 games this season, would also be eligible for the showdown with Atletico.
However, it was confirmed on Wednesday that Barca were unsuccessful with their latest appeal, meaning Lewandowski will miss the match at Estadio Metropolitano.
The ban does not cover Copa del Rey or Supercopa de Espana ties, so Lewandowski will also sit out the LaLiga meetings with Getafe and Girona before the end of January.
Speaking last month, Lewandowski questioned the severity of the punishment and said it would be "painful" to watch three matches from the stands.
Lewandowski also insisted the gesture that landed him in trouble was in fact aimed at Xavi, not the referee.
The former Borussia Dortmund and Bayern Munich star was not part of Barca's squad for Wednesday's Copa del Rey round-of-32 tie with Intercity.
"Lewandowski's absence on Sunday is a setback and it is unexpected," Xavi said after his side's 4-3 victory against the lower-tier opposition.
"We have no choice but to now obey it as we did when they told us he couldn't play against Espanyol."