Acute graft-versus-host disease (aGvHD) is a life-threatening complication typically occurring within 100 days after allogeneic hematopoietic cell transplantation (allo-HCT). This hypothesis-generating, phase II, prospective, open-label, randomized study (clinicaltrials gov. Identifier: NCT03339297) compared defibrotide added to standard-of-care (SOC) GvHD prophylaxis (defibrotide prophylaxis arm) versus SOC alone (SOC arm) to prevent aGvHD post-transplant. This study estimated incidences of aGvHD and was not statistically powered to assess differences among treatment arms. Patients were randomized 1:1 to defibrotide prophylaxis arm (n=79; median age 57 years; range, 2-69 years) or SOC arm (n=73; median age 56 years; range, 2-72 years). Patient demographics in the two arms were similar except for conditioning regimen type (myeloablative: defibrotide, 76% vs. SOC, 61%) and stem cell source for allo-HCT (bone marrow: defibrotide, 34% vs. SOC, 26%). In the intent-to-treat primary endpoint analysis, the cumulative incidence of grade B-D aGvHD at day 100 post-transplant was 38.4% in the defibrotide prophylaxis arm versus 47.1% in the SOC arm (difference: –8.8%, 90% confidence interval [CI]: –22.5 to 4.9). The difference noted at day 100 became more pronounced in a subgroup analysis of patients who received antithymocyte globulin (defibrotide: 30.4%, SOC: 47.6%; difference: –17.2%; 90% CI: –41.8 to 7.5). Overall survival rates at day 180 post-transplant were similar between arms, as were the rates of serious treatment-emergent adverse events (defibrotide: 42%, SOC: 44%). While the observed differences in endpoints between the two arms were not substantial, these results suggest defibrotide prophylaxis may add a benefit to currently available SOC to prevent aGvHD following allo-HCT without adding significant toxicities.