Abstract Cytomegalovirus reactivation (CMVR) after allogeneic hematopoietic cell transplantation (HCT) is a frequent complication related to survival outcomes; however, its impact on relapse remains unclear, especially in acute lymphoblastic leukemia (ALL). In this nationwide retrospective study, we included patients with acute myeloid leukemia (AML) and ALL in the first or second complete remission who underwent their first HCT using a pre-emptive strategy for CMVR. Because 90% of cases with CMVR had occurred by day 64 and 90% of cases with grades 2 to 4 acute graft-versus-host disease (GVHD) had occurred by day 58, a landmark point was set at day 65. In landmark analyses, 3793 patients with AML and 2213 patients with ALL who survived without relapse for at least 65 days were analyzed. Multivariate analyses showed that CMVR was associated with a lower incidence of relapse in both AML (hazard ratio [HR], 0.81; 95% confidence interval [CI], 0.69-0.95; P = .009) and ALL (HR, 0.81; 95% CI, 0.66-0.99; P = .045). These findings were confirmed when CMVR was used as the time-dependent covariate. Moreover, our study suggests that the protective effect of CMVR on relapse was independent of acute GVHD. A post-hoc subgroup analysis of combined AML and ALL showed that CMVR had a mild antileukemia effect without effect modification, in contrast to the impact of CMVR on NRM. Our findings may provide important implications for strategies used for CMV prophylaxis after HCT.