Low vitamin D, commonly assessed as serum 25-hydroxyvitamin D (25OHD), is associated with the development of many age-related chronic diseases. A positive relationship exists between elevated 25OHD and muscle synthesis, strength, power, and decreased body fat in elderly individuals. However, these findings have not been consistently reported in younger healthy populations. The purpose of this study was to investigate the relationship between 25OHD and measures of body size, composition, metabolism, and physical fitness in a young physically active population. Thirty-nine subjects (20 men, 19 women; aged 23 ± 0.7 years) reported 6 times for testing. Blood was collected to determine 25OHD. Primary outcomes included the following: body mass index (BMI) and percent body fat (dual x-ray absorptiometry); resting metabolic rate; maximal oxygen uptake (V[Combining Dot Above]O2max); power output (Wingate); and muscular strength (8 repetition maximum for bench press, upright row, and leg extension and flexion exercises). Our analysis included all participants, and subgroup analyses for individuals with suboptimal 25OHD concentration below 35 ng·mL ("low"; n = 20, 25.97 ± 1.97 ng·mL) or equal to and above 35 ng·mL ("high"; n = 19, 44.15 ± 2.17 ng·mL). Twenty subjects in this study had serum levels of 25OHD below 35 ng·mL. There was a significant positive relationship between V[Combining Dot Above]O2max and serum 25OHD and a negative relationship between BMI and serum 25OHD. These data suggest that vitamin D deficiency is prevalent even in a young physically active population in the southern United States and that there was a positive relationship between a measure of cardiovascular fitness and serum 25OHD, and a negative relationship between serum 25OHD and BMI.