Aim: The objective of this study was to evaluate the efficacy of prolotherapy when treating individuals with lateral epicondylitis through a systematic review and meta-analysis. Methods: The search for articles was carried out in electronic databases including PUBMED, CENTRAL, WEB OF SCIENCE, SCIELO and Google Scholar, published up to July 2021. We used the following keywords: prolotherapy OR proliferation therapy OR hypertonic dextrose injections AND tennis elbow OR lateral epicondylitis. The effectiveness was expressed as mean difference or standardized mean difference ((d) and 95% CI). Major results: Nine clinical trials that used prolotherapy in the treatment of lateral epicondylitis were included. In the pooled analysis, prolotherapy was effective in pain control in the medium (d = -0.85, 95% CI -1.29 to -0.41, p (z) 0.0001) and long terms (d = -1.05, 95% CI -2.06 to -0.03, p (z) 0.04). It was also effective in improving function in the medium term (d = -1.21, 95% CI -1.64 to -0.78, p (z) 0.00001). Conclusions: Prolotherapy was effective for reducing pain in the medium and long terms, as well as for improving function in the medium term, in individuals with lateral epicondylitis. However, the risk of bias of the studies caused that the quality of evidence was moderate; more studies with a low risk of bias are necessary to corroborate the efficacy of prolotherapy in patients with lateral epicondylitis.