BACKGROUND: Whether low plasma 25-hydroxyvitamin D concentrations cause osteoporotic fractures is unclear. We tested the hypothesis that low plasma 25-hydroxyvitamin D concentrations are associated with increased risk of osteoporotic fractures using a Mendelian randomization analysis. METHODS: We genotyped 116 335 randomly chosen white Danish persons aged 20-100 years in 2 population-based cohort studies for plasma 25-hydroxyvitamin D decreasing genotypes in CYP2R1 (rs117913124 and rs12794714), DHCR7 (rs7944926 and rs11234027), GEMIN2 (rs2277458), and HAL (rs3819817); 35 833 had information on plasma 25-hydroxyvitamin D. We assessed risk of total, osteoporotic, and anatomically localized fractures from 1981 through 2017. Information on fractures and vital status was obtained from nationwide registries. RESULTS: During up to 36 years of follow-up, we observed 17 820 total fractures, 10 861 osteoporotic fractures, and 3472 fractures of hip or femur. Compared with individuals with 25-hydroxyvitamin D ≥ 50nmol/L, multivariable adjusted hazard ratios (95% CIs) for total fractures were 1.03 (0.97-1.09) for individuals with 25-49.9 nmol/L, 1.19 (1.10-1.28) for individuals with 12.5-24.9 nmol/L, and 1.39 (1.21-1.60) for individuals with 25-hydroxyvitamin D < 12.5 nmol/L. Corresponding hazard ratios were 1.07 (1.00-1.15), 1.25 (1.13-1.37), and 1.49 (1.25-1.77) for osteoporotic fractures and 1.09 (0.98-1.22), 1.37 (1.18-1.57), and 1.41 (1.09-1.81) for fractures of hip or femur, respectively. Hazard ratios per 1 increase in vitamin D allele score, corresponding to 3.0% (approximately 1.6 nmol/L) lower 25-hydroxyvitamin D concentrations, were 0.99 (0.98-1.00) for total fractures, 0.99 (0.97-1.00) for osteoporotic fractures, and 0.98 (0.95-1.00) for fractures of hip or femur. CONCLUSIONS: Low plasma 25-hydroxyvitamin D concentrations were associated with osteoporotic fractures; however, Mendelian randomization analysis provided no evidence supporting a causal role for vitamin D in the risk for osteoporotic fractures.