Juventus confirmed the 33-year-old will not be able to take his place in the back line for Wednesday's crunch Group D game, with Barca three points clear of Juve at the top of the pool.
Chiellini is among five Juventus players nominated for a place in the 2017 UEFA Team of the Year, along with Gianluigi Buffon, Paulo Dybala, Alex Sandro and Miralem Pjanic.
In his absence, Mehdi Benatia, Benedikt Howedes and Daniele Rugani are the alternative options for Massimiliano Allegri as a defensive partner for Chiellini's fellow veteran Andrea Barzagli.
UPDATE: Giorgio Chiellini is out of tonight's match after picking up a muscle injury to his left thigh. #JuveFCB— JuventusFC (@juventusfcen) November 22, 2017
Juventus will also be without Federico Bernardeschi for the game against Barca, the winger missing the match after suffering a muscle problem.
But Barzagli and goalkeeper Buffon are expected to return to the Juve team after missing Sunday's 3-2 league defeat at Sampdoria in the wake of Italy's World Cup play-off disappointment against Sweden.