Juventus on Monday were given a 10-point deduction in Serie A after a revision of their initial 15-point penalty inflicted on the club over illicit transfer activity.
The new sanction, announced by the Italian Football Federation's (FIGC) appeals court, severely dents Juventus' hopes of qualifying for next season's Champions League, pushing them out of the top four with two matches remaining this season.
Juventus have competed in Europe's elite competition in each of the past 11 seasons dating back to 2012.
In April, Italy's highest sporting court, the Sports Guarantee Board, revoked Juve's 15-point penalty inflicted by the FIGC's appeals court and sent the case for a new judgement.
It asked the appeals court to revise punishments handed down to the club and some key figures, including former vice-chairman Pavel Nedved who was acquitted on Monday.
Juventus said they "reserve the right" to appeal the latest decision to the Sports Guarantee Board.
"While not ignoring the need for urgency, which Juventus has never shied away from during the proceedings, it is emphasised that these are facts that still have to be evaluated by a judge," the Turin giants said on Twitter.
Long bans given to former chairman Andrea Agnelli, ex-CEO Maurizio Arrivabene and sporting directors Federico Cherubini and Fabio Paratici were upheld in April.
But Juventus' hopes that the points deduction would be completely cancelled were quashed on Monday.
Massimiliano Allegri's men, who were second in the top flight, fall to seventh in the table.
Juve are now five points behind fourth-placed AC Milan and host the losing Champions League semi-finalists in their penultimate match of the campaign this weekend.
Juve then visit Udinese in their final match of the season on June 4.
The 'Old Lady' had a chance to move within two points of Milan when they took on Empoli shortly after the points deduction was announced on Monday.
But they slipped to a surprising 4-1 defeat to cap a miserable day for the two-time European champions.
The decision by the FIGC and the result at Empoli also mean that Lazio, who replaced Juve in second place, have qualified for the Champions League group stage for the first time since 2020 and only the second time since 2007.
Inter Milan, who moved into third, Atalanta and Roma have all also jumped one place in the Serie A standings.
Juventus are also embroiled in criminal proceedings over their accounting practices with 12 current and former key club figures including Agnelli potentially facing trial.
According to Italian news agencies, a high court hearing on October 26 will decide whether any eventual trial will be held in Turin, where the investigation has taken place, or in either Milan or Rome.