1-Bromopropane (1-BrP) induces dose- and time-dependent reproductive organ toxicity and reduced sperm motility in rodents. The contribution of cytochrome P4502E1 (CYP2E1) to both 1-BrP metabolism and the induction of male reproductive toxicity was investigated using wild-type (WT) and Cyp2e1^−/− mice. In gas uptake inhalation studies, the elimination half-life of [1,2,3-¹³C]-1-BrP was longer in Cyp2e1^−/− mice relative to WT (3.2 vs. 1.3 h). Urinary metabolites were identified by ¹³C nuclear magnetic resonance. The mercapturic acid of 1-bromo-2-hydroxypropane (2OHBrP) was the major urinary metabolite in WT mice, and products of conjugation of 1-BrP with glutathione (GSH) were insignificant. The ratio of GSH conjugation to 2-hydroxylation increased 5-fold in Cyp2e1^−/− mice relative to WT. After 1-BrP exposure, hepatic GSH was decreased by 76% in WT mice vs. 47% in Cyp2e1^−/− mice. Despite a 170% increase in 1-BrP exposure in Cyp2e1^−/− vs. WT mice, sperm motility in exposed Cyp2e1^−/− mice did not change relative to unexposed matched controls. This suggests that metabolites produced through CYP2E1-mediated oxidation may be responsible for 1-BrP-induced sperm toxicity. Both 1-BrP and 2OHBrP inhibited the motility of sperm obtained from WT mice in vitro. However, only 2OHBrP reduced the motility of sperm obtained from Cyp2e1^−/− mice in vitro, suggesting that conversion of parent compound to 2OHBrP within the spermatozoa may contribute, at least in part, to reduced motility. Overall, these data suggest that metabolism of 1-BrP is mediated in part by CYP2E1, and activation of 1BrP via this enzyme may contribute to the male reproductive toxicity of this chemical.