Objective <p>This systematic review and meta-analysis compared single- versus double-layer uterine closure at the time of cesarean delivery.</p> Data sources <p>MEDLINE, EMBASE, Scopus, ClinicalTrials.gov, and Cochrane Central Register of Controlled Trials were searched from inception until May 2024.</p> Study eligibility criteria <p>We included only randomized controlled trials (RTSs) that compared single-layer versus double-layer uterine closure at the time of cesarean delivery.</p> Study appraisal and synthesis methods <p>Outcomes were analyzed using a random-effects model. Results are expressed as risk differences. The assessment of the risk of bias was performed by using the Risk of Bias 2 tool. The primary outcome was the incidence of scar defects (i.e., niche) at 6&#xa0;months after delivery. The secondary outcomes were incidence of scar defects at 6&#xa0;weeks and 3&#xa0;months.</p> Results <p>A total of 18 studies were identified by the systematic review; 11 RCTs involving 6,058 participants were included in the meta-analysis. There is no statistical difference between single-layer and double-layer uterine closure of cesarean delivery incision regarding the incidence of uterine scar defect at six weeks. Single-layer closure showed a significantly lower incidence of niche after three months (RD = − 0.02 (− 0.06, 0.02); <i>I</i>² = 81%, <i>p</i> &lt; 0.01), and six months (RD = − 0.11, CI − 0.15, − 0.07, I² = 91%, <i>p</i> &lt; 0.01).</p> Conclusions <p>Single-layer uterine closure at the time of cesarean delivery resulted in a lower uterine scar defects after three and six months compared to double-layer uterine closure.</p> Systematic review registration <p>PROSPERO, Unique identifier: CRD42024552495.</p>