Anxiety can impact overall performance and persistence in college. Student response systems (SRSs), real-time active-learning technologies used to engage students and gauge their understanding, have been shown to elicit anxiety for some students. Kahoot! is an SRS technology that differs from others in that it involves gamification, the use of gamelike elements. Recent studies have explored the impact of active-learning strategies on student anxiety across different institutions, but there is little known about how Kahoot! impacts student perceived anxiety, especially in comparison with other active-learning strategies. In two complementary yet parallel studies of introductory biology courses at a western research-intensive institution (n = 694) and a southeastern research-intensive institution (n = 60), we measured students’ perceived anxiety. We then explored how students were influenced by nongraded Kahoot! play and other elements of instruction. Using previously developed and course-specific pre- and post-course surveys, we found students at both universities agreed that nongraded Kahoot! play caused less anxiety compared with other pedagogical practices, such as working in small groups or reading the textbook. After playing Kahoot!, lower-performing students demonstrated greater engagement and lower levels of anxiety compared with their peers, suggesting that Kahoot! may be a particularly engaging active-learning strategy for these students.