Maternal hyperhomocysteinemia is detrimental for reproduction, but the effects on embryo quality are unknown. The aim of this study was to investigate whether biomarkers of the homocysteine pathway are associated with in vitro fertilization (IVF) outcome.In a prospective study, we investigated biomarkers of the homocysteine pathway for associations with embryo quality and biochemical pregnancy in women undergoing IVF or intracytoplasmic sperm injection treatment (n = 181). In the treatment cycle, blood and monofollicular fluid samples were collected for determination of folate, cobalamin and total homocysteine (tHcy) concentrations.Of all the women in the study, 67% used folic acid supplements. In blood, a significant correlation was established between high cobalamin and better embryo quality [standardized adjusted regression coefficient: -0.17, 95% confidence interval (CI): -0.30, -0.01]. In monofollicular fluid of non-supplemented women, high cobalamin correlated with better embryo quality (estimate: -0.87; 95% CI: -1.68, -0.06), whereas high tHcy resulted in poor embryo quality (estimate: 1.01; 95% CI: 0.08, 1.95). However, in monofollicular fluid of supplemented women, high tHcy correlated with better embryo quality (estimate: -0.58; 95% CI: -1.12, -0.04). In the total group, a 2-fold increase of monofollicular fluid folate corresponded with a 3.3 times higher chance (95% CI: 1.09, 9.71) of achieving pregnancy.An optimal homocysteine pathway in follicular fluid is associated with a better embryo quality and chance of pregnancy.