The 27-year-old suffered the latest in a series of serious injuries when he damaged the anterior cruciate ligament in his right knee in January.
Rossi recovered in time to be named in Cesare Prandelli's provisional 30-man squad, and was involved in the 0-0 warm-up draw with the Republic of Ireland on Saturday.
However, Rossi suffered the heartbreak of being excluded from Prandelli's final 23-man party for the marquee tournament in Brazil, which was announced on Sunday.
Rossi has since taken to social media website Twitter to deny that his omission was due to concerns over fitness or his psychological mindset.
"Everyone says I'm out of shape: ask anyone the results from the tests during the week and the game. You will be shocked," Rossi wrote.
"Challenges? Fear? What a laugh. But now the important thing is to support Italy. Forza Azzurri."
It marks further disappointment for the American-born forward, after he was overlooked for Italy's 2010 squad, with former manager Marcello Lippi later admitting he made a mistake by not taking him to South Africa.
Italy begin their Group D campaign against England on June 14.
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