Aim To investigate the plausible relationship between the polymorphism of methylenetetrahydrofolate reductase (MTHFR) gene at C677T locus and serum lipid levels and longevity in the long-lived cohort residing in Guangxi Hongshuihe River Basin. Methods Genotyping was performed with PCR-RFLP technique for 505 long-lived Zhuang individuals inhabiting in Guangxi Hongshuihe River Basin whose ages were 90 and above (long-lived group, LG) and 468 ethnic- and geographic-matched healthy mid-aged or elderly controls (non-long-lived group, non-LG). Association analysis were undertaken between MTHFR C677T genotypes and serum lipids (total cholesterol, TC triglyceride, TG high density lipoprotein, HDL low density lipoprotein, LDL). Results The allelic (C and T) and genotypic (CC, CT, and TT) frequencies of the LG and non-LG were 80.2%, 19.8% versus 85.0%, 15.0% and 65.5%, 29.3%, 5.2% versus 73.9%, 22.2%, 3.8%, respectively, which displayed significant differences between the two tested groups, with an overrepresentation of T allele in long-lived females specially, but not in males. On lipid profiles, the levels of TC, TG, LDL in LG are significantly higher than that in the non-LG. After stratifying by MTHFR C677T genotype and gender, the TC, TG, and LDL levels were noted dramatically higher in females but not in males harboring the mutant genotype (CT/TT) than that of the non-T carriers (CC) in the LG. Conclusions Our data suggest that there was a femalespecific association between the MTHFR C677T polymorphism and serum lipid levels and human longevity, which may be one of the molecular genetic basis for the longevity in the Guangxi Hongshuihe River Basin.