by Ling Lu, Derrick A. Bennett, Iona Y. Millwood, Sarah Parish, Mark I. McCarthy, Anubha Mahajan, Xu Lin, Fiona Bragg, Yu Guo, Michael V. Holmes, Shoaib Afzal, Børge G. Nordestgaard, Zheng Bian, Michael Hill, Robin G. Walters, Liming Li, Zhengming Chen, Robert Clarke Background Observational studies have reported that higher plasma 25-hydroxyvitamin D (25[OH]D) concentrations are associated with lower risks of diabetes, but it is unclear if these associations are causal. The aim of this study was to test the relevance of 25(OH)D for type 2 diabetes using genetically instrumented differences in plasma 25(OH)D concentrations. Methods and findings Data were available on four 25(OH)D single nucleotide polymorphisms (SNPs; n = 82,464), plasma 25(OH)D concentrations ( n = 13,565), and cases with diabetes ( n = 5,565) in the China Kadoorie Biobank (CKB). The effects on risk of diabetes were assessed by a genetic score using two 25(OH)D synthesis SNPs ( DHCR7 -rs12785878 and CYP2R1 -rs10741657), with and without the addition of SNPs affecting the transport ( GC/DBP -rs2282679) and catabolism ( CYP24A1 -rs6013897) of 25(OH)D. The CKB results were combined in a meta-analysis of 10 studies for the 2 synthesis SNPs ( n = 58,312 cases) and 7 studies for all 4 SNPs ( n = 32,796 cases). Mean (SD) 25(OH)D concentration was 62 (20) nmol/l in CKB, and the per allele effects of genetic scores on 25(OH)D were 2.87 (SE 0.39) for the synthesis SNPs and 3.54 (SE 0.32) for all SNPs. A 25-nmol/l higher biochemically measured 25(OH)D was associated with a 9% (95% CI: 0%–18%) lower risk of diabetes in CKB. In a meta-analysis of all studies, a 25-nmol/l higher genetically instrumented 25(OH)D concentration was associated with a 14% (95% CI: 3%–23%) lower risk of diabetes ( p = 0.01) using the 2 synthesis SNPs. An equivalent difference in 25(OH)D using a genetic score with 4 SNPs was not significantly associated with diabetes (odds ratio 8%, 95% CI: −1% to 16%, lower risk, p = 0.07), but had some evidence of pleiotropy. A limitation of the meta-analysis was the access only to study level rather than individual level data. Conclusions The concordant risks of diabetes for biochemically measured and genetically instrumented differences in 25(OH)D using synthesis SNPs provide evidence for a causal effect of higher 25(OH)D for prevention of diabetes.