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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 &#177; 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&#174; immunochemiluminometric analyzer. The results showed a negative association of 1,25(OH)₂D with BMI, LMI and BMD (&#946; = &#8722;0.274, R² = 0.075, <i>p</i> = 0.019; &#946; = &#8722;0.268, R² = 0.072, <i>p</i> = 0.022; and &#946; = &#8722;0.325, R² = 0.105, <i>p</i> = 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.