Isner started the US Open Series with a disappointing loss in the second round of the 2016 Rogers Cup to Ryan Harrison. Isner rebounded by reaching his first final of the season and his fourth consecutive final at the Atlanta Open, losing in straight sets to Nick Kyrgios. Isner skipped the Summer Olympics due to it lacking ranking points and prize money, electing to focus on the summer hardcourt tournaments of the US Open Series. After Isner lost in straight sets in the second round to Milos Raonic and Steve Johnson reached the quarterfinals in Cincinnati, Isner lost the title of the top-ranked American man for the first time in three years. Although he regained it in the following week, Johnson took it back again following the 2016 US Open, after Isner was unable to defend his fourth-round points, losing in the third round to Kyle Edmund in four sets.