Purpose. To determine vitamin D metabolites and vitamin D receptor (VDR) single-nucleotide polymorphisms (SNPs) relationships with physical performance. Methods. In 1205 men and 322 women (94.8% white Caucasian, 22.0 ± 2.8 years) commencing military training, we measured: serum vitamin D metabolites (25-hydroxyvitamin D (25(OH)D) and 24,25-dihydroxyvitamin D (24,25(OH)2D) by high-performance liquid chromatography tandem mass spectrophotometry, and 1,25-dihydroxyvitamin D (1,25(OH)2D) by immunoassay); VDR SNPs (rs2228570, rs4516035, and rs7139166 by polymerase chain reaction genotyping); and endurance performance by 2.4 km run, muscle strength by maximal dynamic lift, and muscle power by maximal vertical jump. Results. Serum 25(OH)D was negatively associated with 2.4 km run time and positively associated with muscle power (β = –12.0 and 90.1), 1,25(OH)2D was positively associated with run time and negatively associated with strength and muscle power (β = 5.6, –1.06, and –38.4), and 24,25(OH)2D was negatively associated with run time (β = –8.9; P < 0.01), after controlling for age, sex, smoking, alcohol, physical activity, time outdoors, season, and BMI. Vitamin D metabolites (25(OH)D, 1,25(OH)2D, and 24,25(OH)2D) together explained variances of 5.0% in run time, 0.7% in strength, and 0.9% in muscle power (ΔF P < 0.001). All performance measures were superior with low 1,25(OH)2D:24,25(OH)2D ratio (P < 0.05). VDR SNPs were not associated with physical performance (ΔF P ≥ 0.306). Conclusion. Vitamin D metabolites accounted for a small portion of variance in physical performance. Associations between vitamin D metabolites and run time were the most consistent. VDR SNPs explained no variance in performance. Greater conversion of 25(OH)D to 24,25(OH)2D, relative to 1,25(OH)2D (i.e., low 1,25(OH)2D:24,25(OH)2D ratio), was favourable for performance, indicating 24,25(OH)2D may have a role in optimising physical performance