Background: The association between vitamin D and acute graft-versus-host disease (GvHD) remains controversial, especially for patients receiving myeloablative conditioning. Methods: We measured pre-transplantation plasma vitamin D (25-hydroxyvitamin D₃ + D₂) levels by competitive electrochemiluminescence in plasma samples from 116 adult patients who underwent a myeloablative allogeneic transplantation at Rigshospitalet, Copenhagen between July 2015 and August 2018. Results: The median (Q1, Q3) pre-transplantation plasma vitamin D level was 64 (47, 85) nmol/L (normal range: 50–160 nmol/L). Vitamin D insufficiency (<50 nmol/L) and moderate deficiency (<25 nmol/L) were observed in 29% and 8% of patients, respectively. No patients had a severe deficiency (<12 nmol/L). Pre-transplantation vitamin D levels were slightly higher in patients who later developed grade II–IV acute GvHD (mean difference: 8.1 nmol/L), but the 95% confidence interval [CI] encompassed clinically insignificant differences (CI: −2.2, 19.2 nmol/L). From multivariable logistic regression, we found that a patient with a pre-transplantation vitamin D level of 85 nmol/L (Q3) had 1.5 times higher odds of grade II–IV acute GvHD than a patient with a level of 47 nmol/L (Q1; CI of odds ratio: 0.84, 2.7; adjusted for patient age, donor type, use of anti-thymocyte globulin, and use of 12 Gy total-body irradiation). Patients with pre-transplantation vitamin D insufficiency (N = 34) had a cumulative incidence of grade II–IV acute GvHD similar to that of patients with vitamin D sufficiency (26% [CI: 11%, 42%] versus 35% [CI: 25%, 46%], respectively). Conclusions: Our data did not support an association between pre-transplantation vitamin D levels or vitamin D insufficiency and acute GvHD in adult patients receiving myeloablative conditioning.