Summary Context In observational studies, low serum 25‐hydroxyvitamin D (25‐OHD) concentration is associated with an increased risk of type 2 diabetes mellitus (DM). Increasing serum 25‐OHD may have beneficial effects on insulin resistance or beta‐cell function. Cross‐sectional studies utilizing suboptimal methods for assessment of insulin sensitivity and serum 25‐OHD concentration provide conflicting results. Objective This study examined the relationship between serum 25‐OHD concentration and insulin resistance in healthy overweight individuals at increased risk of cardiovascular disease, using optimal assessment techniques. Methods A total of 92 subjects (mean age 56·0, SD 6·0 years), who were healthy but overweight (mean body mass index 30·9, SD 2·3 kg/m2), underwent assessments of insulin sensitivity (two‐step euglycaemic hyperinsulinaemic clamp, HOMA2‐IR), beta‐cell function (HOMA2%B), serum 25‐OHD concentration and body composition (DEXA). Results Mean total 25‐OHD concentration was 32·2, range 21·8–46·6 nmol/l. No association was demonstrated between serum 25‐OHD concentration and insulin resistance. Conclusions In this study using optimal assessment techniques to measure 25‐OHD concentration, insulin sensitivity and body composition, there was no association between serum 25‐OHD concentration and insulin resistance in healthy, overweight individuals at high risk of developing cardiovascular disease. This study suggests the documented inverse association between serum 25‐OHD concentration and risk of type 2 DM is not mediated by a relationship between serum 25‐OHD concentration and insulin resistance.