Openers Murali Vijay and Shikhar Dhawan have instead been picked as the Men in Blue look to put their faith in youth over experience.
Wicketkeeper-batsman Dinesh Karthik has been recalled after almost three years, while India also include all-rounder Irfan Pathan.
The selectors seem to prefer Rohit Sharma in the middle-order to Yuvraj Singh, who made his comeback last year after fighting a rare form of lung cancer.
Gambhir's omission is even more of a surprise after his usual opening partner Virender Sehwag was left out of the 30-man squad.
India open the tournament against South Africa in Cardiff on June 6.
India's ICC Champions Trophy squad: Mahendra Dhoni (capt & wk), Murali Vijay, Shikhar Dhawan, Dinesh Karthik (wk), Virat Kohli, Suresh Raina, Rohit Sharma, Ravindra Jadeja, Ravichandran Ashwin, Amit Mishra, Irfan Pathan, Umesh Yadav, Bhuvneshwar Kumar, Ishant Sharma, Vinay Kumar