The purpose of this study was to examine the effects of vitamin D supplementation on inflammatory biomarkers in overweight and obese adults participating in a progressive resistance exercise training program. Twenty-three (26.1 ± 4.7 years) overweight and obese (BMI 31.3 ± 3.2 kg/m2) adults were randomized into a double-blind vitamin D supplementation (Vit D 4,000 IU/day; female 5, male 5) or placebo (PL, female 7; male 6) intervention trial. Both groups performed 12 weeks (3 days/week) of progressive resistance exercise training (three sets of eight exercises) at 70-80% of one repetition maximum. Whole-blood lipopolysaccharide (LPS)-stimulated tumor necrosis factor (TNF) α production as well as circulating C-reactive protein (CRP), TNFα, interleukin 6 (IL-6), and alanine aminotransferase (ALT) were assessed at baseline and after the 12-week intervention. No main effects of group or time were detected for circulating CRP, TNFα, IL-6, and ALT. As expected, when PL and Vit D groups were combined, there was a significant correlation between percent body fat and CRP at baseline (r = 0.45, P = 0.04), and between serum 25OHD and CRP at 12 weeks (r = 0.49, P = 0.03). The PL group had a significant increase in 25 μg/ml LPS + polymixin B-stimulated TNFα production (P = 0.04), and both groups had a significant reduction in unstimulated TNFα production (P < 0.05) after the 12-week intervention. Vitamin D supplementation in healthy, overweight, and obese adults participating in a resistance training intervention did not augment exercise-induced changes in inflammatory biomarkers.