Isner reached the fourth round at the BNP Paribas Open for the third year in a row, where he lost to Kei Nishikori. He was then upset early at the 2016 Miami Open by Tim Smyczek. Despite missing most of the clay season, only playing the U.S. Men's Clay Court Championships and the Geneva Open, Isner reached the fourth round of the 2016 French Open for only the second time in his career, losing to eventual finalist Andy Murray in straight sets. Isner's season continued with early losses in both grass tournaments he played, losing to Gilles Müller at the 2016 Aegon Championships in addition to a marathon match against Jo-Wilfried Tsonga at the 2016 Wimbledon Championships.