Obesity and sarcopenia are major causes of morbidity and mortality among seniors. Vitamin D deficiency is very common especially among seniors and has been associated with both muscle health and obesity. This study investigated if 25-hydroxyvitamin D (25(OH)D) status is associated with body composition and insulin resistance using baseline data of a completed RCT among relatively healthy community-dwelling seniors (271 seniors age 60+ years undergoing elective surgery for unilateral total knee replacement due to osteoarthritis). Cross-sectional analysis compared appendicular lean mass index (ALMI: lean mass kg/height m²) and fat mass index (FMI: fat mass kg/height m²) assessed by DXA and insulin resistance between quartiles of serum 25(OH)D concentration using multivariable linear regression adjusted for age, sex, smoking status, physical activity, and body mass index (BMI). Participants in the lowest serum 25(OH)D quartile (4.7⁻17.5 ng/mL) had a higher fat mass (9.3 kg/m²) compared with participants in the third (8.40 kg/m²; Q3 = 26.1⁻34.8 ng/mL) and highest (8.37 kg/m²; Q4 = 34.9⁻62.5 ng/mL) quartile ( = 0.03). Higher serum 25(OH)D quartile status was associated with higher insulin sensitivity ( = 0.03) and better beta cell function ( = 0.004). Prevalence of insulin resistance tended to be higher in the second compared with the highest serum 25(OH)D quartile (14.6% vs. 4.8%, = 0.06). Our findings suggest that lower serum 25(OH)D status may be associated with greater fat mass and impaired glucose metabolism, independent of BMI and other risk factors for diabetes.