Summary Background & aims The aim of this study is to further clarify the role of plasma 25(OH)D concentration after a weight-lowering nutritional intervention on body composition, blood pressure and inflammatory biomarkers in overweight/obese middle-aged subjects. Methods This longitudinal research encompassed a total of 50 subjects 57.26 (5.24) year, who were under a 15% energy restricted diet for 4 weeks. Anthropometric and body composition variables, blood routine, inflammatory markers as well as 25(OH)D were analysed. Results Circulating 25(OH)D levels 12.13(±17.61%) increased while anthropometric, body composition, routine blood markers as well as the concentration of TNF-α, C-reactive protein and Lp-PLA2 were significantly reduced after the intervention. Multiple linear regression analyses evidenced that Δ25(OH)D increase was linked to the decrease in weight, adiposity, SBP and IL-6 levels. Moreover, a relationship was found between Δ25(OH)D, Δfat mass ( r  = −0.405; p  = 0.007), ΔSBP ( r  = −0.355; p  = 0.021) and ΔIL-6 ( r  = −0.386; p  = 0.014). On the other hand, a higher increase in 25(OH)D was accompanied by reductions in weight, BMI, SBP, IL-6 and an increase in bone mineral concentration ( p  < 0.05). Interestingly, higher levels of 25(OH)D at the endpoint, showed a significantly higher decrease in weight, BMI and total fat mass. Conclusions The increase in plasma 25(OH)D level is linked with the decrease in SBP and adiposity in middle-aged subjects after a weight-loss intervention. Therefore, 25(OH)D assessment is a potential marker to be accounted in metabolic measures related to blood pressure, adiposity and inflammation in obesity management. Trial registration www.clinicaltrials.gov (NCT01596309).