Ulster hooker Best, 34,  is suffering from a stomach bug so Munster's Niall Scannell, 24, will win his first cap at the Stadio Olimpico.
Leinster's James Tracy, who earned his first cap against Canada in November, has been promoted to the bench.
Best's regular Ireland understudy Sean Cronin has been ruled out of the entire Six Nations by a hamstring injury.
Skipper Best missed Friday's captain's run in Rome after feeling unwell overnight.
The Ireland management expressed optimism on Friday that Best would recover sufficiently to lead the side but he was ruled out of the game just over an hour before kick-off.
Vice-captain Jamie Heaslip will now skipper the side in Rome as Best's run of 51 successive Six Nations games comes to an end.
Ireland are aiming to regroup in Rome after their surprise opening defeat by Scotland last weekend.
The Irish line-out misfired during the first half at Murrayfield and there will be fears that Scannell's late call-up could put the visitors under further pressure in the crucial set-piece.
Italy clinched their first Six Nations victory over an under-strength Ireland in Declan Kidney's final season in charge in 2013 but Joe Schmidt's side have won four subsequent contests against the Azzurri.
Ireland skipper Rory Best has been ruled out of Saturday's Six Nations game against Italy because of illness.