Vitamin D deficiency is a worldwide health problem that, in addition to its well-known negative effects on musculoskeletal health, has been related to a wide range of acute and chronic age-related diseases. However, little is known about the association of body composition with the active, hormonal form of vitamin D, 1,25-dihydroxyvitamin D plasma levels (1,25(OH)2D). Therefore, the aim of this study was to investigate the association of 1,25(OH)2D with body composition including lean and fat body mass as well as bone mineral density (BMD) in middle-aged sedentary adults. A total of 73 (39 women) middle-aged sedentary adults (53.7 ± / 5.1 years old) participated in the current study. We measured weight and height, and we used dual energy X-ray absorptiometry to measure lean body mass, fat body mass and BMD. Body mass index (BMI), lean mass index (LMI), and fat mass index (FMI) were calculated. 1,25(OH)2D was measured using a DiaSorin Liaison® / immunochemiluminometric analyzer. The results showed a negative association of 1,25(OH)2D with BMI, LMI and BMD (&beta / = &minus / 0.274, R2 = 0.075, p = 0.019 / &beta / = &minus / 0.268, R2 = 0.072, p = 0.022 / and &beta / = &minus / 0.325, R2 = 0.105, p = 0.005, respectively), which persisted after controlling for age and sex. No significant differences in 1,25(OH)2D across body weight status were observed after controlling for the same covariates. In summary, our results suggest that 1,25(OH)2D could be negatively associated with BMI, LMI and BMD whereas no association was found with FMI in middle-aged sedentary adults.