Rubisco activase (RCA) facilitates the removal of inhibitors from the Rubisco active site and is essential for plant growth at atmospheric CO2. Here we report the phenotypes of gene edited RCA mutants in the C4 grass Setaria viridis, which has two forms of RCA, RCAα and RCAβ, encoded by separate genes. In Setaria, only the β form accumulates at normal temperatures, with the α form being induced following exposure to heat. Inactivating the RCAβ gene (βKO) abolished accumulation of RCA at 25°C and significantly reduced photosynthesis. These plants required elevated CO2 for survival, while inactivating RCAα (αKO) did not affect RCAβ accumulation, photosynthesis or growth. Neither mutant exhibited altered Rubisco content or activation when grown at 25°C and elevated CO2. When βKO plants were transferred from elevated to ambient CO2 and 40°C, RCAα induction supported growth for approximately 7 days, although plants were smaller with fewer tillers compared to WT and αKO plants. The ability of a small amount of RCAα to support growth in ambient CO2 at 40°C is consistent with our results showing that its in vitro ATPase activity is more thermotolerant than RCAβ and suggests that RCAα may help C4 plants cope with certain heat stress conditions.