BACKGROUND: Cross-sectional studies suggest an association of 25-hydroxyvitamin D with exacerbations in patients with COPD but longitudinal evidence from cohort studies is scarce. Our aim was to assess the association of serum 25-hydroxyvitamin D with exacerbations and mortality in primary care patients with COPD. METHODS: We included 356 COPD patients (GOLD stages II-IV, free of exacerbations for ≥4 weeks) from a prospective cohort study in Dutch and Swiss primary care settings in the main analysis where we assessed the association of 25-hydroxyvitamin D with (centrally adjudicated) exacerbations and mortality using negative binomial and Cox regression, respectively. RESULTS: Baseline mean serum 25-hydroxyvitamin D concentration was 15.5 ng/dl (SD 8.9) and 274 patients (77.0%) had 25-hydroxyvitamin D deficiency (<20 ng/dl). Compared to patients with severe 25-hydroxyvitamin D deficiency (<10 ng/dl, n=106 [29.8%]), patients with moderately deficient (10-19.99 ng/dl, n=168 [47.2%]) and insufficient (20-29.99 ng/dl, n=58 [16.3%]) concentrations had the same risk for exacerbations (incidence rate ratios of 1.01 [95% CI 0.77-1.57] and 1.00 [0.62-1.61], respectively). In patients with desirable concentrations (>30 ng/dl, n=24 [6.7%]) the risk was lower, although not statistically significantly (0.72 [0.37-1.42]). Including patients with vitamin D supplements, using different cut-offs for 25-hydroxyvitamin D or competing risk models did not materially change the results. We did not find a statistically significant association of 25-hydroxyvitamin D concentration with mortality. CONCLUSION: This longitudinal study in a real-world COPD population that carefully minimized misclassification of exacerbations and the influence of confounding did not show an association of 25-hydroxyvitamin D with exacerbations and mortality. STUDY REGISTRATION ClinicalTrials.gov (NCT00706602).