We used an established mediation analysis to investigate the role of adiposity in the relation between serum 25(OH)D with markers of inflammation and glucose and insulin metabolism. We used data from National Health and Nutrition Examination Survey (2005–2010), to evaluate the associations between serum 25(OH)D and markers of insulin resistance (IR) or inflammation, and whether these associations are mediated by adiposity factors. Analysis of co-variance and conceptual causal mediation analysis were conducted taking into consideration the survey design and sample weights. BMI was found to have significant mediation effects (to varied extent) on the associations between serum 25(OH)D and CRP, apo-B, fasting glucose, insulin, HOMA-IR, HOMA-B and HbA1c (all P < 0·05). Both WC and apVAT were also found to partly mediate the associations between serum 25 25(OH)D with CRP, FBG, HbA1c, TAG and HDL-cholesterol (all P < 0·05). These findings support the importance of optimising 25(OH)D status in conditions with abnormal adiposity (i.e. obesity) and treatments for the prevention of cardiometabolic diseases affecting adipose tissue metabolism (i.e. weight loss).