GLUT4 is a major mediator of glucose removal from the circulation and a key regulator of whole-body glucose homeostasis. Recent studies in south Indian populations revealed that haplotypes of the GLUT4 gene associated with type 2 diabetes. A total of 734 middle aged apparently healthy Japanese men were recruited from two separate occupational cohorts from Kanagawa and Kyoto. Participants were genotyped for GLUT4 variants, rs5418 (A/G) and rs2654185 (C/A), and association with HbA1c level was analyzed. The HbA1c value was determined by JDS method which is 0.4% lower than NGSP value. The G allele carrier of rs5418 and A allele carrier of rs2654185 associated with significantly higher HbA1c level (AG + GG vs. AA carriers; 5.2 ± 0.8 vs. 4.9 ± 0.4, P < 0.002, and AA + AC vs. CC; 5.2 ± 0.9, vs. 4.9 ± 0.4, P < 0.002, respectively). G allele, AG + GG genotype of rs5418 and A allele, AA + AC genotype of rs2654185 showed a significant association with higher HbA1c (β = 0.215, P = 0.026; β = 0.215, P = 0.026; β = 0.190, P = 0.042; β = 0.190, P = 0.042, respectively). These two SNPs are in high linkage disequilibrium (LD) of r(2) = 0.67. In haplotype analysis, four haplotypes were estimated. HbA1c is significantly higher in the most frequent GA haplotype compared with the second frequent AC haplotype (5.2% vs. 5.1%, P = 0.004). Genetic variations, rs5418 and rs2654185 in GLUT4 gene are associated with HbA1c level in Japanese men.