Background It has been suggested that overweight and obese individuals have an increased risk to develop vitamin D deficiency, commonly associated with poor muscle performance. The relationship among fat mass, vitamin D status, and skeletal muscle is still debated. Aims To evaluate the effects of the combination of hypovitaminosis D and overweight on muscle mass and strength, and physical performance in post-menopausal women. Methods In this cross-sectional study, we recruited post-menopausal women referring to a physiatric outpatient service for the management of osteoporosis over a 36-month period. We compared four groups: (1) normal weight with hypovitaminosis D; (2) overweight with normal serum 25(OH)D 3 ; (3) overweight with hypovitaminosis D; and (4) normal weight with normal serum 25(OH)D 3 (control group). Outcome measures were: appendicular lean mass-to-BMI ratio; hand grip strength; and short physical performance battery. Results We analysed 368 women (mean aged 67.2 ± 7.8 years): 95 normal weight with hypovitaminosis D, 90 overweight with normal levels of 25(OH)D 3 , 96 overweight with hypovitaminosis D, and 87 normal weight with normal levels of 25(OH)D 3 . Overweight women with hypovitaminosis D had a significant risk of reduced muscle mass (OR 5.70; p  < 0.001), strength (OR 12.05; p  < 0.001), and performance (OR 5.84; p  < 0.001) compared to controls. Normal weight women with hypovitaminosis D had only a greater risk of an impairment of muscle strength (OR 7.30; p  < 0.001) and performance (OR 3.16; p  < 0.001). Discussion According to our findings, both hypovitaminosis D and overweight should be investigated in post-menopausal women because of their negative effects on skeletal muscle mass and function. Conclusions This study demonstrated that hypovitaminosis D is associated to impaired muscle function and its combination with overweight might lead also to muscle wasting in a cohort of post-menopausal women.