Epidemiological studies suggest a relationship between total 25-hydroxyvitamin D [25(OH)D], adiposity, and metabolic traits. The bioavailability of 25(OH)D is regulated by the albumin, vitamin D binding protein (VDBP), and variants of the GC gene. Therefore, it is not clear if bioavailable or free 25(OH)D offer additional benefits compared to total 25(OH)D when estimating the magnitude of these associations. Our aim was to evaluate the association between 25(OH)D (total, free and bioavailable) with adiposity and metabolic traits. This was a cross-sectional study of 1904 subjects from the Health Workers Cohort Study from Mexico. Free and bioavailable 25(OH)D were calculated based on VDBP and albumin determinations, using a formula adjusted for the GC gene diplotypes. Adiposity and metabolic traits were measured with standardized procedures. Free and bioavailable 25(OH)D levels correlated with total 25(OH)D, r = 0.71 and 0.70, respectively (p < 0.001). Total, bioavailable and free 25(OH)D levels were negatively associated with the adiposity marker (visceral adiposity index) and metabolic traits (metabolic syndrome, type 2 diabetes, triglycerides, triglycerides/HDL-c ratio, and triglycerides/glucose index) in multivariate regression models (ORs = 0.73 to 0.96). Our findings suggest that free and bioavailable 25(OH)D do not offer additional advantages over total 25(OH)D regarding its association with adiposity and several metabolic traits in Mexican adults.