Background/Aims: An association of genetic variants of homocysteine (Hcy) metabolic genes with type 2 diabetes mellitus (T2DM) has been reported. The objective of the present study was to investigate the relationship between the genetic variants in Hcy metabolism-related genes and plasma Hcy levels and T2DM susceptibility in Han Chinese. Methods: A total of 774 patients with T2DM and 500 healthy individuals were recruited. Single-nucleotide polymorphism was determined by standard methods. Results: The Hcy-increasing allele score was positively associated with plasma Hcy levels in both T2DM patients and healthy subjects (r = 0.171 and 0.247, respectively). Subjects with the genotype CC of MTHFR (rs1801131) had a significantly higher likelihood of T2DM compared with subjects with the AA or AA+AC genotypes (OR = 1.93 for CC vs. AA, p = 0.041; OR = 3.13 for CC vs. AA+AC, p = 0.017, respectively). Subjects with the genotype AA of the MTHFD variant (rs2236225) had a significantly lower likelihood of T2DM compared with subjects with the GG or GG+GA genotypes (OR = 0.36 for AA vs. GG, p = 0.027; OR = 0.36 for AA vs. GG+GA, p = 0.017, respectively). In addition, the genotype CT+TT of the PEMT (rs4646356) variants displayed a significant association with an increased risk of T2DM (OR = 1.52 for CT+TT vs. CC, p = 0.042). Conclusions:MTHFR rs1801131 C allele and PEMT rs4646356 T allele were associated with a high risk of T2DM in these Han Chinese.