Objective: The aim of this study was to determine the effect of preoperative surgical antibiotic prophylaxis (SAP) with additional intraoperative redosing compared to single-dose preoperative surgical antibiotic prophylaxis on the incidence of surgical site infections (SSI). Summary Background Data: Preoperative SAP is standard care for the prevention of SSI. During long surgical procedures, additional intraoperative redosing of SAP is advised, but there is great variability in redosing strategies and compliance rates. Methods: We performed a systematic search of MEDLINE (PubMed), Embase, CINAHL and CENTRAL on June 25th, 2021 according to PROSPERO registration CRD42021229035. We included studies that compared the effect of preoperative SAP with additional intraoperative redosing to single dose preoperative SAP (no redosing) on SSI incidence in patients undergoing any type of surgery. Two researchers performed data appraisal and extraction of summary data independently. Meta-analyses were stratified per study type. We used a generic inverse variance random-effects model to estimate a pooled odds ratio with corresponding 95% confidence intervals (CIs). Results: We included 2 randomized controlled trials (RCT) and 8 cohort studies comprising of 9470 patients. Pooled odds ratios for SSI in patients receiving intraoperative redosing compared to those without redosing were 0.47 (95% CI: 0.19–1.16. I2 = 36%) for RCTs and 0.55 (95% CI: 0.38–0.79, I2 = 56%) for observational cohorts. There was considerable clinical heterogeneity among antibiotics used and redosing protocols. GRADE-assessment showed overall low certainty of evidence. Conclusion: Intraoperative redosing of SAP may reduce incidence of SSI compared to a single dose preoperative SAP in any type of surgery, based on studies with considerable heterogeneity of antibiotic regimens and redosing protocols.