Vitamin D is associated with skeletal muscle physiology and function and may play a role in intramuscular inflammation, possibly via the vitamin D receptor (VDR). We conducted two studies to examine (1) whether serum 25-hydroxyvitamin D (25OHD) and/or intramuscular VDR protein concentrations are associated with intramuscular interleukin-6 (IL-6) and/or tumor necrosis factor-α (TNFα); and (2) whether 16-week supplementation with vitamin D3 alters intramuscular IL-6 and/or TNFα. Potential-related signaling pathways were also examined. Muscle biopsies of 30 older, mobility-limited adults were obtained at baseline. A subset of 12 women were supplemented with either 4,000 IU/day of vitamin D3 (N = 5) or placebo (N = 7), and biopsies were repeated at 16 weeks. Serum 25OHD was measured, and intramuscular VDR, IL-6, and TNFα gene expressions and protein concentrations were analyzed. Baseline serum 25OHD was not associated with intramuscular IL-6 or TNFα gene expression or protein concentration. Baseline intramuscular VDR protein concentration, adjusted for baseline serum 25OHD, was positively associated with intramuscular IL-6 gene expression (n = 28; p = 0.04), but negatively associated with intramuscular IL-6 protein (n = 18; p = 0.03). Neither intramuscular IL-6 nor TNFα gene expression was different between placebo (n = 7) or vitamin D3 supplementation groups (n = 5) after 16 weeks (p = 0.57, p = 0.11, respectively). These data suggest that VDR is a better predictor than serum 25OHD concentration of intramuscular IL-6 gene and protein expressions. A similar relationship was not observed for TNFα expression. Further, supplementation with 4,000 IU vitamin D3 per day does not appear to affect intramuscular IL-6 or TNFα gene expression after 16 weeks.