Background: Resting metabolic rate (RMR) used to prognosticate and measure the amount of energy required. Vitamin D is known as a new predictor of RMR. The aim of this study is to investigate the relationship between vitamin D effects on RMR in connection with the vitamin D receptor (VDR) and peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) gene expression. Methods: We enrolled 298 overweight and obese adults in this cross-sectional study. Body mass index (BMI), fat mass, fat-free mass, insulin level, visceral fat, and vitamin D status were assessed. RMR was measured by means of indirect calorimetry. The real-time polymerase chain reaction using specific primer pairs for VDR and PGC-1α was performed. Results: There were significant differences in terms of fat free mass, fat percentage, insulin levels, RMR/kg body weight, and RMR/BMI, VDR, and PGC-1α among participants were categorized based on the vitamin D status. But after using general linear model for adjusting, all significant results missed their effectiveness except RMR/kg body weight and VDR. Linear regression analysis used to show the mediatory role of VDR and PGC-1α on the RMR/kg body weight and vitamin D status relationship. Our results showed that VDR had a mediatory effect on the relationship between RMR/kg body weight and vitamin D status (β = 0.38, 95% CI –0.48 to 1.60; β = –1.24, 95% CI –5.36 to 1.70). However, PGC-1α did not affect the relationship between RMR/kg body weight and vitamin D status (β = 0.50, 95% CI = –0.02 to 3.42; β = 0.59, 95% CI 0.14–3.90). Conclusion: Our study showed the mediatory effect of VDR gene expression in the association of 25(OH)2D plasma levels and resting metabolic rate among obese individuals.