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Previous studies have explored associations between betaine and diabetes, but few have considered the effects of genes on them. We aimed to examine associations between serum betaine, methyl-metabolizing genetic polymorphisms and the risk of type 2 diabetes in Chinese adults. This prospective study comprised 1565 subjects aged 40–75 without type 2 diabetes at baseline. Serum betaine was measured by high-performance liquid chromatography tandem mass spectrometry. Genotyping of methyl-metabolizing genes was detected by Illumina ASA-750K arrays. Cox proportional hazards model was used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs). During a median of 8.9 years of follow-up, 213 participants developed type 2 diabetes. Compared with participants in the lowest quartile of serum betaine, those in the highest quartile had lower risk of type 2 diabetes, adjusted HRs (95%CIs) was 0.46 (0.31, 0.69). For methylenetetrahydrofolate reductase (<i>MTHFR</i>) G1793A (rs2274976) and <i>MTHFR</i> A1298C (rs1801131), participants carrying 1793GA + AA and 1298AC + CC had lower risk of type 2 diabetes. Interactions of serum betaine and genotype of <i>MTHFR</i> G1793A and <i>MTHFR</i> A1298C could be found influencing type 2 diabetes risk. Our findings indicate that higher serum betaine, mutations of <i>MTHFR</i> G1793A and A1298C, as well as the joint effects of them, are associated with lower risk of type 2 diabetes.