Low vitamin D and insulin resistance are common in polycystic ovary syndrome (PCOS) and associated with higher inter- and intra-muscular adipose tissue (IMAT). We investigated associations between vitamin D, IMAT and insulin resistance in a cross-sectional study of 40 women with PCOS and 30 women without PCOS, and pre- and post-exercise in a 12-week intervention in 16 overweight participants (10 with PCOS and six without PCOS). A non-classical body mass index (BMI) threshold was used to differentiate lean and overweight women (BMI ≥ 27 kg/m2). Measurements included plasma 25-hydroxyvitamin D (25OHD), insulin resistance (glucose infusion rate (GIR; mg/m2/min), fasting glucose and insulin, and glycated haemoglobin), visceral fat, mid-thigh IMAT (computed tomography) and total body fat (dual-energy X-ray absorptiometry). Women with both PCOS and low 25OHD levels had the lowest GIR (all p