Objective: To test the hypothesis that genetically low 25-hydroxyvitamin D concentrations are associated with increased mortality.

Design: Mendelian randomisation analysis.

Setting: Copenhagen City Heart Study, Copenhagen General Population Study, and Copenhagen Ischemic Heart Disease Study.

Participants: 95 766 white participants of Danish descent from three cohorts, with median follow-up times of 19.1, 5.8, and 7.9 years, genotyped for genetic variants in DHCR7 and CYP2R1 affecting plasma 25-hydroxyvitamin D concentrations; 35 334 also had plasma 25-hydroxyvitamin D measurements. Participants were followed from study entry through 2013, during which time 10 349 died.

Main outcome measures: All cause mortality and cause specific mortality, adjusted for common risk factors for all cause mortality based on the World Health Organization's global health status.

Results: The multivariable adjusted hazard ratios for a 20 nmol/L lower plasma 25-hydroxyvitamin D concentration were 1.19 (95% confidence interval 1.14 to 1.25) for all cause mortality, 1.18 (1.09 to 1.28) for cardiovascular mortality, 1.12 (1.03 to 1.22) for cancer mortality, and 1.27 (1.15 to 1.40) for other mortality. Each increase in DHCR7/CYP2R1 allele score was associated with a 1.9 nmol/L lower plasma 25-hydroxyvitamin D concentration and with increased all cause, cancer, and other mortality but not with cardiovascular mortality. The odds ratio for a genetically determined 20 nmol/L lower plasma 25-hydroxyvitamin D concentration was 1.30 (1.05 to 1.61) for all cause mortality, with a corresponding observational multivariable adjusted odds ratio of 1.21 (1.11 to 1.31). Corresponding genetic and observational odds ratios were 0.77 (0.55 to 1.08) and 1.13 (1.03 to 1.24) for cardiovascular mortality, 1.43 (1.02 to 1.99) and 1.10 (1.02 to 1.19) for cancer mortality, and 1.44 (1.01 to 2.04) and 1.17 (1.06 to 1.29) for other mortality. The results were robust in sensitivity analyses.

Conclusions: Genetically low 25-hydroxyvitamin D concentrations were associated with increased all cause mortality, cancer mortality, and other mortality but not with increased cardiovascular mortality. These findings are compatible with the notion that genetically low 25-hydroxyvitamin D concentrations may be causally associated with cancer and other mortality but also suggest that the observational association with cardiovascular mortality could be the result of confounding.