Low-dose post-transplant cyclophosphamide (PTCy) in conjunction with anti-thymocyte globulin (ATG) appears as a potentially effective graft-versus-host disease (GVHD) prevention strategy in haploidentical hematopoietic cell transplant (haplo-HCT). Our study aims to assess the efficacy of this regimen. We extended our prospective study in patients treated with low-dose PTCy (14.5 mg/kg on days 3 and 4) in ATG/granulocyte colony-stimulating factor (G-CSF)-based regimen and compared the results to the contemporary cohort of patients without low-dose PTCy (ATG cohort). Both study cohort and control are transplanted from maternal donor or collateral relatives. We identified 239 consecutive patients (ATG-PTCy cohort = 114; ATG cohort = 125). All patients but one in ATG cohort achieved myeloid engraftment by day 30 post-HCT. We found that both the cumulative incidence of 100-day grade III-IV aGvHD and non-relapse-mortality (NRM) in the ATG-PTCy cohort was significantly reduced than that in the ATG group (5% vs 18%; P = 0.003; and 6% vs 15%; P= 0.045); the 2-year cumulative incidences of relapse and overall survival were comparable between the two cohorts (13% vs 14%; P = 0.62; and 83% vs 77%; P = 0.18, respectively). Furthermore, GVHD-free, relapse-free survival (GRFS) was significantly improved in the ATG-PTCy arm (63% vs 48%; P = 0.039). In multivariate analysis, the joint treatment resulted in lower grade II-IV acute GVHD (HR 0.58; P = 0.036), grade III-IV aGvHD (HR 0.28; P = 0.006), chronic GVHD (HR 0.60; P = 0.047), NRM (HR 0.26; P = 0.014), and higher GRFS (HR 0.59; P = 0.021) but slower myeloid and platelet recovery (HR 0.29 and 0.30; both P < 0.001). These results suggested that ATG/PTCy (low-dose) can reduce both acute and chronic GVHD as compared with standard ATG-based prophylaxis using maternal donor or collateral relatives at particular high GVHD risk.