Background: Glutathione S-transferases (GSTs) metabolize drugs and xenobiotics. Yet despite high protein sequence homology, expression of π--class GSTs, the most abundant of the enzymes, varies significantly between species. In mouse liver, hepatocytes exhibit high mGstp expression, while in human liver, hepatocytes contain little or no hGSTP1 mRNA or hGSTP1 protein. π--class GSTs are known to be critical determinants of liver responses to drugs and toxins: when treated with high doses of acetaminophen, mGstp1/2 mice suffer marked liver damage, while mGstp1/2-/- mice escape liver injury. Methodology/Principal Findings: To more faithfully model the contribution of π--class GSTs to human liver toxicology, we introduced hGSTP1, with its exons, introns, and flanking sequences, into the germline of mice carrying disrupted mGstp genes. In the resultant hGSTP1+mGstp1/2-/- strain, π--class GSTs were regulated differently than in wild-type mice. In the liver, enzyme expression was restricted to bile duct cells, Kupffer cells, macrophages, and endothelial cells, reminiscent of human liver, while in the prostate, enzyme production was limited to basal epithelial cells, reminiscent of human prostate. The human patterns of hGSTP1 transgene regulation were accompanied by human patterns of DNA methylation, with bisulfite genomic sequencing revealing establishment of an unmethylated CpG island sequence encompassing the gene promoter. Unlike wild-type or mGstp1/2-/- mice, when hGSTP1+mGstp1/2-/- mice were overdosed with acetaminophen, liver tissues showed limited centrilobular necrosis, suggesting that π--class GSTs may be critical determinants of toxin-induced hepatocyte injury even when not expressed by hepatocytes. Conclusions: By recapitulating human π--class GST expression, hGSTP1+mGstp1/2-/- mice may better model human drug and xenobiotic toxicology. ©2011 Vaughn et al.