Speaking at the European Commission office in Italy, Letta said he found his chances to take the top job near “impossible”.
“I consider it highly improbable, if not impossible, that there could be another Italian other than Mario Draghi at the top of the European institutions,” Letta was quoted in Il Sole 24 Ore as saying.
Draghi is currently the president of the European Central Bank (ECB) and the former governor of the Bank of Italy (Banca d’Italia).
Speculation has been rife in recent weeks over who will take over from Barroso, who has served two terms as the Commission president and will step down later this year.
German Chancellor Angela Merkel has backed Jean-Claude Juncker, the former prime minister of Luxembourg, although has faced opposition from other EU leaders such as the UK’s David Cameron.
The debate surrounding who will take up the post has been described by Austrian Chancellor Werner Faymann as a “regrettable spectacle”.
With Letta suggesting he is out of the running, another politician said to be a possible candidate is Swedish Prime Minister Fredrik Reinfeldt.