Venipuncture is a common invasive clinical procedure, and pain management during puncture has been of interest to healthcare professionals. The purpose of this systematic review and meta-analysis was to evaluate the efficacy and safety of the Valsalva maneuver (VM) for the relief of venipuncture pain in children and adults. PubMed, Embase, Web of Science, Cochrane Library, CNKI, Wanfang, VIP database, and CBM were searched from inception to December 2023 for all available randomized controlled trials (RCTs) that evaluated the impact of VM on venipuncture. Two reviewers independently performed study selection, data extraction, and risk of bias assessment. Continuous variables were analyzed by mean differences (MD) or standardized mean differences (SMD), whereas dichotomous variables were analyzed by risk ratios (RR). A total of 22 studies involving 1740 participants were included. The pooled results showed that VM relieved pain intensity during venipuncture in children (SMD = -0.89, 95% CI = -1.47 to -0.30,