Elevated plasma total homocysteine (tHcy) is an independent risk factor for ischemic heart disease and stroke. Epidemiological studies reveal that men have higher tHcy levels than women but the mechanism underlying this sex-dependent difference is unknown. One route for intracellular disposal of homocysteine is catalyzed by cystathionine -synthase (CBS). Renal function is known to be an important determinant of tHcy and in this study, we demonstrate that renal CBS expression and activity in mice diminished ~2-fold after castration, whereas ovariectomization was without effect. The higher renal CBS activity in males (22.7 ± 3.1 mmol cystathionine h^-1 kg kidney^-1) versus females (8.4 ± 3.4 mmol cystathionine h^-1 kg kidney^-1, P10^-6) in C57Bl/6J mice was associated with lower plasma tHcy levels in males vs. females, and this difference was exacerbated in Cbs+/- mice (7.7 ± 1.9 µmole L^-1 in males versus 13.8 ± 6.4 µmole L^-1 in females, P = 0.005). Surprisingly, mammals exhibit a diversity of regulatory patterns for kidney CBS, with females exhibiting lower CBS activity in mice, higher in rats and humans and being indistinguishable from males in rabbit, hamster and guinea pig. Our data suggest that testosterone-dependent regulation of human CBS in kidney may contribute to sex-dependent differences in homocysteine transsulfuration.