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Objective To provide a systematic review and meta-analysis of prospective, population-based cohort studies on the association of serum 25-hydroxyvitamin D (25(OH)D) and all-cause mortality. Methods Relevant studies were identified by systematically searching Medline, EMBASE and ISI Web of Knowledge. Reported hazard ratios (HRs) for 25(OH)D categories were recalculated employing comprehensive trend estimation from summarized dose-response data and pooled in a random effects model meta-analysis. Results Overall, 12 original studies were included in the review and meta-analysis comprising 32,142 mainly elderly study participants with measured 25(OH)D of whom 6921 died during follow-up. An inverse association between 25(OH)D levels and all-cause mortality was found in all but two studies that was statistically significant in several of the individual studies. In meta-analysis, 25(OH)D levels were significantly inversely associated with all-cause mortality with a pooled HR of 0.92 (95% confidence interval: 0.89–0.95) for a 20 nmol/l increase in 25(OH)D levels. Conclusion In this meta-analysis of prospective, population-based cohort studies, a 20 nmol/l increase in 25(OH)D levels was associated with an 8% lower mortality in the general elderly population. This agrees with results from meta-analyses on randomized controlled trials that found a decrease in mortality with vitamin D3 supplementation of a comparable magnitude.