Previous studies have shown that methylenetetrahydrofolate reductase (MTHFR) gene to be a genetic risk factor for the susceptibility to ischemic stroke. The aim of this case-control study was to investigate whether the polymorphisms of MTHFR C677T were associated with the susceptibility to ischemic stroke in a southern Chinese Hakka population. In this study, a total of 1967 ischemic stroke patients and 2565 controls of Chinese Hakka ethnicity were recruited. The MTHFR C677T polymorphisms were genotyped by polymerase chain reaction (PCR) amplification and microarray method. The risk of ischemic stroke was estimated by logistic regression analysis. The frequencies of CC, CT, and TT genotypes were 52.67% versus 55.63%, 40.31% versus 38.52%, and 7.02% versus 5.85% in patients with ischemic stroke versus controls, respectively. The frequency of T allele was significantly higher in ischemic stroke patients (27.17%) than in controls (25.11%) (P = .026, odds ratio [OR] 1.113, 95% confidence interval [CI] 1.013-1.223). The homozygous TT genotype in the ischemic stroke patients was associated with increased risk (P = .049, OR 1.132, 95% CI 1.001-1.281) when compared with the controls after adjustment for age and sex. The positive association was only found in dominant model without adjustment for age and sex (P = .047, OR 1.127, 95% CI 1.002-1.268). Also, the carrier status of the MTHFR T allele was identified as an independent risk factor for the development ischemic stroke even after the adjustment for conventional risk factors (P = 0.047, OR 1.109, 95% CI 0.964-1.225). Our results provide evidence that variants of MTHFR C677T gene may influence the risk of developing ischemic stroke in a southern Chinese Hakka population. Further studies are needed to confirm this association, which will promote the development of strategies for prevention and treatment of ischemic stroke in our study population.