The 25-year-old Azzurri forward had been hoping to make his return to Primera Division action in the coming weeks but he broke down in training on Friday morning
The 25-year-old forward had been nearing a return to competitive action after first injuring his right knee during his club's Liga loss to Real Madrid at the tail end of October.
However, Villarreal announced on Friday afternoon that the player tore his ACL again during a training session earlier in the day.
Rossi now requires another operation on his knee and is expected to be out for a further six months.
The news will come as a major blow for Azzurri boss Cesare Prandelli, who had been hoping that Rossi would make his comeback before the season's end and thus give the coach an extra striking option for the country's Euro 2012 campaign.
The injury could also scupper the former Manchester United man's hopes of a summer transfer, with the player having been heavily linked with a return to Italy in recent months.