Mitochondrial DNA (mtDNA) variants have been implicated in many diseases including diabetes mellitus. To explore whether these genetic variants contribute to the susceptibility for type 2 diabetes mellitus (T2DM) in a Chinese population, a total of 184 T2DM cases and 279 matched healthy controls were recruited. PCR restriction fragment length polymorphism (PCR-RFLP) analysis and DNA sequencing were used to determine the variants of mtDNA (including T16189C, G3316A, T3394C, A14693G, A3243G and C1310T). Some of them were further analyzed by mfold or tRNA-scan-SE software. A homoplastic A14693G, for the first time, was found in 4 of 184 Chinese cases, the frequency of A14693G and T3394C was 2.17% and 2.72%, respectively, in patients but not in the controls. Secondary structure prediction revealed that there were obvious conformational changes in T3394C mutant ND1 versus wild type and A14693G mutant tRNA(Glu) protein versus wild type, providing additional clues to the disease pathogenesis although A3243G and C1310T mutations were not detected in any patients in the two groups. The 16189 variant among type 2 diabetes was more prevalent than in controls (36.9% versus 28.7%, P=0.039), and stepwise multiple regression analysis showed that the 16189 variant was an independent factor contributing to HOMA-IR (R(2)=0.043, P=0.037). Our results suggest that the mutations of T3394C and A14693G may contribute to genetic predisposition to T2DM, with the T16189C variant being associated with insulin resistance.