Background: Prolotherapy, which stimulates the healing of loosened ligaments and tendons, is a cost-effective and safe treatment modality for chronic musculoskeletal pain. Its benefits may be affected by injection protocols, comparative regimens, and evaluation scales. The aim of this study was to determine the effectiveness of dextrose prolotherapy as a long-term treatment for chronic musculoskeletal pain. Methods: Medline, Embase, Cochrane Central, KoreaMed, and KMbase databases were searched for studies published up to March 2019. We included randomized controlled trials which compared the effect of dextrose prolotherapy with that of other therapies such as exercise, saline, platelet-rich plasma, and steroid injection. The primary outcome was pain score change during daily life. Results: Ten studies involving 750 participants were included in the final analysis. Pain scores from 6 months to 1 year after dextrose prolotherapy were significantly reduced compared to saline injection (standardized mean difference [SMD] –0.44; 95% confidence interval [CI] –0.76 to –0.11, P = 0.008) and exercise (SMD –0.42; 95% CI –0.77 to –0.07, P = 0.02). Prolotherapy yielded results similar to platelet-rich plasma or steroid injection, that it showed no significant difference in pain score. Conclusions Dextrose prolotherapy is more effective in the treatment of chronic pain compared to saline injection or exercise. Its effect was comparable to that of platelet-rich plasma or steroid injection. Adequately powered, homogeneous, and longer-term trials are needed to better elucidate the efficacy of prolotherapy.