Objective · To investigate the association between polymorphisms in methylenetetrahydrofolate reductase (MTHFR), serum homocysteine (HCY) concentration and outcome of assistant reproductive technology (ART). Methods · A retrospective study was conducted in 285 infertile couples who underwent ART. Sperm quality, serum HCY concentration and MTHFR genotype of the males in these couples were collected. According to the results of MTHFR C677T and serum HCY of males, they were divided into four groups, i.e, TT+high HCY, TT+normal HCY, NOT TT+high HCY, and NOT TT+normal HCY (as control). Results · There was no significant difference in the frequency distribution of oligoasthenozoospermia of four groups (P>0.05). The fertilization rates were significantly lower in patients with high plasma HCY levels (P<0.05). The rates of effective embryo in NOT TT+high HCY group were significantly lower (P<0.05). Accumulated pregnancy rates and early abortion rates were not significantly different among four groups (P>0.05). Conclusion · High serum HCY of males may affect outcome of ART. The detection of MTHFR gene polymorphisms helps to remind supplement folic acid in time.