Purpose This study aimed to determine vitamin D metabolites and vitamin D receptor (VDR) single-nucleotide polymorphism (SNP) relationships with physical performance. Methods In 1205 men and 322 women (94.8% White Caucasian, 22.0 ± 2.8 yr) 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 body mass index. 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 (ΔFP ≥ 0.306). Conclusions 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 favorable for performance, indicating 24,25(OH)2D may have a role in optimizing physical performance.