Vitamin D is essential for bone health and has immunomodulatory properties. Most pediatric patients are vitamin D insufficient (<30 ng/mL) before hematopoietic stem cell transplantation (HSCT). Standard supplementation strategies fail to achieve vitamin D sufficiency in the acute post-transplantation period, and there are scarce data to support optimal vitamin D supplementation in this patient population. This study aimed to evaluate whether a single, oral, weight-based ultra-high dose of vitamin D (Stoss dosing) was more effective than standard supplementation to achieve pre-HSCT vitamin D sufficiency and reduce the incidence of HSCT-related complications (acute graft-versus-host disease, veno-occlusive disease, and/or transplant-associated thrombotic microangiopathy) that are associated with immune-mediated endothelial damage. Secondary endpoints examined the immunomodulatory properties of vitamin D. We conducted a nonrandomized controlled clinical trial of Stoss-dosed vitamin D in pediatric patients receiving HSCT. The study prospectively enrolled 33 patients, 29 of whom successfully received Stoss-dosed vitamin D and were compared to 136 patients in a historical control. Patient characteristics were compared using Fisher's exact test or t-test. The one-sided Fisher's exact test was used for cohort comparison of the primary endpoints. Logistic regression was used to examine the association between patient-specific factors and total 25-hydroxy vitamin D (25-OHD) levels and the compiled HSCT complications. In the Stoss cohort, 97% (n = 28/29) of patients achieved pre-HSCT vitamin D sufficiency compared to 67% (n = 10/15) of patients in the historical control who were on standard supplementation at the time the total 25-OHD level was assessed (P = .013). The mean total 25-OHD level in the Stoss cohort was significantly higher than patients in the historical control who received standard supplementation (72.2 ng/mL versus 35.8 ng/mL, P < .001). Nine patients in the Stoss cohort maintained vitamin D sufficiency throughout the first 100 days after HSCT, and the remaining 19 patients maintained sufficiency for a median of 63 days (range 6-105 days) from the Stoss dose. Patients receiving Stoss-dosed vitamin D developed a lower combined incidence of HSCT-related complications than the historical control (25% [n = 7/28] versus 42% [n = 57/136], P = .055). After Stoss dosing, immunophenotyping studies found a significant decrease in subsets of CD8+ T cells and mononuclear cells (P = .040 and.013, respectively), and, in a subset of cells, larger decreases in phosphoprotein expression were seen with greater increases in total 25-OHD levels. Inflammatory cytokines did not change significantly after Stoss dosing. Stoss dosing is therefore a safe and effective approach to maintain vitamin D sufficiency in the immediate post-HSCT period and may be associated with decreased HSCT-related complications. Randomized studies are warranted to further investigate the efficacy of Stoss-dosed vitamin D to improve bone health and reduce complications in pediatric patients receiving HSCT.