Abstract Background An inverse association between vitamin D status and cardiometabolic risk has been reported but this relationship may have been affected by residual confounding from adiposity and physical activity due to imprecise measures of these variables. We aimed to investigate the relationship between serum 25-hydroxyvitamin D (25(OH)D) and cardiometabolic risk factors, with adjustment for objectively-measured physical activity and adiposity. Methods This was a population-based cross-sectional study in 586 adults in Cameroon (63.5% women). We assessed markers of glucose homoeostasis (fasting blood glucose (BG), 2 h post glucose load BG, HOMA-IR)) and computed a metabolic syndrome score by summing the sex‐specific z‐scores of five risk components measuring central adiposity, blood pressure, glucose, HDL cholesterol and triglycerides. Results Mean±SD age was 38.3 ± 8.6 years, and serum 25(OH)D was 51.7 ± 12.5 nmol/L. Mean 25(OH)D was higher in rural (53.4 ± 12.8 nmol/L) than urban residents (50.2 ± 12.1 nmol/L), p  = 0.002. The prevalence of vitamin D insufficiency (<50 nmol/L) was 45.7%. There was an inverse association between 25(OH)D and the metabolic syndrome score in unadjusted analyses (β = −0.30, 95% CI −0.55 to −0.05), which became non-significant after adjusting for age, sex, smoking status, alcohol intake and education level. Serum 25(OH)D was inversely associated with fasting BG (−0.21, −0.34 to −0.08)), which remained significant after adjustment for age, sex, education, smoking, alcohol intake, the season of data collection, BMI and physical activity (−0.17, −0.29 to −0.06). There was an inverse association of 25(OH)D with 2-h BG (−0.20, −0.34 to −0.05) and HOMA-IR (−0.12, −0.19 to −0.04) in unadjusted analysis, but these associations became non-significant after adjustment for potential confounders. Conclusion Vitamin D insufficiency was common in this population. This study showed an inverse association between vitamin D status and fasting glucose that was independent of potential confounders, including objectively measured physical activity and adiposity, suggesting a possible mechanism through insulin secretion.