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The updated meta‐analysis was conducted to further verify the effect of varicocele on sperm DNA damage, supplying clinicians and researchers with high‐grade evidence. The sperm DNA damage was evaluated by DNA fragmentation index (DFI), associated with the male fertility capability tightly. PubMed, Web of Science and Cochrane Library were searched extensively for eligible studies with the search terms: varicocele, sperm DNA and sperm DNA damage. Finally, a total of 12 studies were included in our meta‐analysis with a total of 845 patients diagnosed with varicocele and 2,377 healthy controls. A statistical difference of DFI between varicocele patients and healthy controls was found after pooling the data ((Standardised mean difference) SMD: 1.40, 95%CI: 0.83–1.98, p <.0001), using the random effect model. We conducted subgroup analysis according to study region (Brazil and Other countries), detection methods of DFI (TUNEL, Comet, and SCSA), sample size (<50 and >50) and age (<30 and >30 years), based on substantial heterogeneity among eligible studies. The stability of pooled results was verified by sensitivity analysis. All these statistical analyses were conducted using Stata version 16.0. In conclusion, patients diagnosed with clinical varicocele had higher DFI than healthy controls, which means varicocele could impair sperm DNA, consequently the fertility potential of affected men. [ABSTRACT FROM AUTHOR]