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 males, 19 females; 23 ± 0.7 years old) reported 6 times for testing. Blood was drawn to determine 25OHD. Primary outcomes included: body mass index (BMI), percent body fat (dual x-ray absorptiometry, DXA); resting metabolic rate; maximal oxygen uptake (VO2max); power output (Wingate); and muscular strength (eight repetition maximum for bench press, upright row and leg extension and flexion exercises). Our analysis included all participants, and sub-group analyses for individuals with sub-optimal 25OHD below (LOW; n = 20, 25.97 ± 1.97 ng/mL) or equal to and above (HIGH; n = 19, 44.15 ± 2.17 ng/mL) 35 ng/mL. Twenty subjects in this study had serum levels of 25OHD below 35 ng/mL. There was a significant positive relationship between VO2max 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.