Submariners spend prolonged periods submerged without sunlight exposure and may benefit from vitamin D supplementation to maintain vitamin D status. The primary objective of this study was to determine the efficacy of daily vitamin D supplementation on maintenance of 25-hydroxyvitamin D (25(OH)D) during a 3-month submarine patrol. Submariners were randomly divided into three groups: placebo (n = 16), 1,000 IU/day (n = 20), or 2,000 IU/day (n = 17). Anthropometrics, self-reported dietary calcium and vitamin D intake, serum markers of vitamin D and bone metabolism, and peripheral quantitative computed tomography (pQCT) parameters of the tibia were determined before and after the patrol. Prior to departure, 49 % of the subjects were vitamin D insufficient (<50 nmol/L). Following the patrol, 25(OH)D increased in all groups (p < 0.001): 3.3 ± 13.1 (placebo), 4.6 ± 11.3 (1,000 IU/day), and 13 ± 14 nmol/L (2,000 IU/day). The changes in 25(OH)D levels were dependent upon the baseline concentration of 25(OH)D and body mass (p < 0.001). Osteocalcin increased by 38 % (p < 0.01), and pQCT analyses revealed small, yet significant increases in indices of tibial structure and strength (p < 0.05) that were independent of supplementation. These data suggest that vitamin D status was low prior to the patrol, and the subsequent changes in vitamin D status were dependent on the baseline 25(OH)D levels and body mass. Furthermore, short-term skeletal health does not appear to be negatively affected by 3 months of submergence in spite of a suboptimal response to vitamin D supplementation.