Background Gamification refers to the use of game elements in nongame contexts. The use of gamification to change behaviors and promote physical activity (PA) is a promising avenue for tackling the global physical inactivity pandemic and the current prevalence of chronic diseases. However, there is no evidence of the effectiveness of gamified interventions with the existence of mixed results in the literature. Objective The aim of this systematic review and meta-analysis is to evaluate the effectiveness of gamified interventions and their health care potential by testing the generalizability and sustainability of their influence on PA and sedentary behavior. Methods A total of 5 electronic databases (PubMed, Embase, Scopus, Web of Science, and the Cochrane Central Register of Controlled Trials) were searched for randomized controlled trials published in English from 2010 to 2020. Eligibility criteria were based on the components of the participants, interventions, comparators, and outcomes framework. Studies were included when they used gamified interventions in daily life with an active or inactive control group and when they assessed a PA or sedentary behavior outcome. We conducted meta-analyses using a random-effects model approach. Sensitivity analyses, influence analyses, and publication bias analyses were performed to examine the robustness of our results. Results The main meta-analysis performed on 16 studies and 2407 participants revealed a small to medium summary effect of gamified interventions on PA behavior (Hedges g=0.42, 95% CI 0.14-0.69). No statistical difference among different subgroups (adults vs adolescents and healthy participants vs adults with chronic diseases) and no interaction effects with moderators such as age, gender, or BMI were found, suggesting good generalizability of gamified interventions to different user populations. The effect was statistically significant when gamified interventions were compared with inactive control groups, such as waiting lists (Hedges g=0.58, 95% CI 0.08-1.07), and active control groups that included a nongamified PA intervention (Hedges g=0.23, 95% CI 0.05-0.41). This suggests that gamified interventions are not only efficient in changing behavior but also more effective compared with other behavioral interventions. The long-term effect (measured with follow-up averaging 14 weeks after the end of the intervention) was weaker, with a very small to small effect (Hedges g=0.15, 95% CI 0.07-0.23). Conclusions This meta-analysis confirms that gamified interventions are promising for promoting PA in various populations. Additional analyses revealed that this effect persists after the follow-up period, suggesting that it is not just a novelty effect caused by the playful nature of gamification, and that gamified products appear effective compared with equivalent nongamified PA interventions. Future rigorous trials are required to confirm these findings.