Female-to-male (F→M) sex-mismatched allogeneic hematopoietic stem cell transplantation (HSCT) is known to increase the risk of graft-versus-host disease (GVHD). We evaluated the impact of donor-recipient sex mismatch on GVHD incidence and assessed the efficacy of combined low-dose antithymocyte globulin (ATG) (2mg/kg) and post-transplant cyclophosphamide (PTCy) as GVHD prophylaxis compared to calcineurin inhibitor-methotrexate/mycophenolate mofetil (CNI-MTX/MMF). We retrospectively analyzed 861 HSCT recipients, with acute myeloid leukemia as the predominant indication (82%). Among the cohort, 39% of transplants were sex-mismatched (M→F: 26%, F→M: 13%), while 61% were sex-matched (M→M: 42%, F→F: 19%). The primary outcomes were cumulative incidences of acute and chronic GVHD, relapse, and nonrelapse mortality (NRM). F→M HSCT were associated with higher rates of grades II-IV acute GVHD at day +100 (42.2% versus 27.0%, hazard ratio [HR]: 1.54; P < .01) and chronic GVHD at 2 years (54.2% versus 43.4%, HR: 1.33; P = .05). In the overall cohort, ATG-PTCy was associated with a reduced risk of grades III-IV acute GVHD (HR: .42; P < .01) and chronic GVHD (HR: .22; P < .001) compared to CNI-MTX/MMF, without an increased risk of relapse (HR: .86; P = .39) or NRM (HR: .59; P = .35). A subgroup multivariable analysis of F→M recipients (n = 114) confirmed a reduced risk of grade II-IV (HR: .48; P = .05), grades III-IV acute GVHD (HR: .25; P = .04), and chronic GVHD (HR: .33; P < .01) with ATG-PTCy. F→M sex mismatch is associated with increased GVHD risk after HSCT. The combination of low-dose ATG and PTCy may help reduce GVHD in this high-risk group without an increase in disease relapse or NRM.