Isner, 29, finished the 2014 season with a 39-20 win-loss record on the ATP World Tour, winning titles in Auckland and Atlanta.
Gimelstob joins Isner's camp after working as a broadcaster for the Tennis Channel, and following an 11-year professional career that saw him climb as high as number 63 in the world.
Two-time mixed doubles major winner Gimelstob retired in 2007, and is undertaking his first coaching role - and he will continue his commentary duties on tour.
Isner said he was in need of a change of voice.
"I'm really excited to start working with Justin. He has so much experience within the tennis business and I've long respected his knowledge of the sport," Isner said.
"I think it will be great for me to have a new perspective helping me with my game.
"I had a great run with Mike [Sell] and feel I really learned a lot from him throughout the course of our coaching relationship.
"I just thought it was time to try something new, and with Justin I believe I will have a strong, and passionate new voice on my team.
"In spending some time with Justin throughout the fall I have full confidence that he has a clear vision for my game and an understanding of what I need to do in order to play my best tennis."
Gimelstob said he was excited to begin a partnership with Isner, a one-time US Open quarter-finalist.
"I have the highest regard for the level of commitment coaching a player of John's pedigree entails," Gimelstob said.
"I am excited for the new challenge, and look forward to pursuing it with a player that I believe in so much.
"I have had a great relationship with John for years and think we will communicate really well as player and coach."
The top-ranked American will begin his 2015 season by defending his Heineken Open title in New Zealand.