Vitamin D, a hormone involved in bone and calcium homeostasis, has been shown to have potent immunomodulatory effects. We performed a retrospective cohort analysis to evaluate whether monohydroxyvitamin D levels before allogeneic hematopoietic SCT (HSCT) correlate with the risk of GVHD. Fifty-three patients who underwent myeloablative HSCT were studied. Vitamin D levels were measured in serum samples obtained before HSCT. The median 25-hydroxyvitamin vitamin D level was 21.9 ng/mL (7.8-45.7). The cumulative incidence (CI) of grades II-IV acute GVHD at 100 days was 53.1% in patients with vitamin D<25, versus 33.3% in patients with vitamin D≥25 ng/mL (P = 0.13). The CI of chronic GVHD (cGVHD) at 2 years in patients with vitamin D<25 was 63.8%, compared with 23.8% in patients with vitamin D≥25 ng/mL (P = 0.009). Similarly, the 2 year CI of extensive cGVHD was significantly greater in patients with vitamin D<25 compared with those with vitamin D≥25 ng/mL (54.5% versus 14.3%, P = 0.005). In a multivariable competing risk model, low pre-transplant vitamin D levels remained a significant factor associated with cGVHD (hazard ratio = 5.26, P = 0.02). Our results demonstrate that vitamin D deficiency before HSCT is associated with an increased risk of cGVHD.