Background: Eccentric exercise is thought to improve joint flexibility, but the size of the effect is not known. We aimed to quantify the overall effect of eccentric exercise on joint flexibility in adults. Design: Systematic review, meta-analysis. Data sources: AMED, CINAHL, MEDLINE, EMBASE, SportDiscus. Participants: Adults. Intervention: Eccentric exercise compared to no intervention or to a different intervention. Outcome measures: Joint range of motion or muscle fascicle length. Data extraction and synthesis: Descriptive data of included trials and estimates of effect sizes were extracted. Standardised mean differences (SMD) of range of motion or fascicle length outcomes were meta-analysed using random effects models. Overall quality of evidence was assessed using the GRADE scale. Results: 32 trials (1122 participants, 108 lost to follow-up) were included in the systematic review. The mean (SD) PEDro score was 5.2 (1.3). Four trials reported insufficient data for meta-analysis. Data from 27 trials (911 participants, 82 lost to follow-up) were meta-analysed. Eccentric exercise improved joint flexibility in adults (pooled random effects Hedges' g SMD = 0.54, 95% CI 0.34 to 0.74). The true effect size is different across studies and 50% of the variance in observed effects is estimated to reflect variance in true effects rather than sampling error (I2 = 50%, Q = 67.6, d.f. = 34, p = 0.001). Overall quality of evidence ranged from `low┐ to `high┐. Conclusion: Eccentric exercise improves joint flexibility in adults. The overall standardised mean effect of eccentric exercise was moderately large, and the narrow width of the 95% confidence interval indicates the effect was estimated with good precision. Registration: Open Science Foundation (https://osf.io/mkdqr); PROSPERO registration CRD42020151303.