Aristolochic acid I (AAI), the most prominent component of aristolochic acids and found in nearly all aristolochic herbs, has been demonstrated significant nephrotoxicity. In this study, an acute nephrotoxicity model of AAI mice was established by a single dose injection of AAI. It was observed that there are differences of the sensitivity to AAI nephrotoxicity in female and male mice, with male mice exhibiting nephrotoxic effects even at lower doses. After the administration of estradiol (E2), serum levels of creatinine and urea nitrogen in male mice were observed to decrease. We used UPLC-MS/MS to determine the pharmacokinetics and renal tissue distribution of AAI and its metabolite aristololactam I (ALI). It was found that AAI had a longer plasma half-life in female mice, while the content of ALI in renal tissue of male mice was much higher than that in female. The administration of E2 was found to extend the half-life of AAI and reduce the levels of ALI in the kidneys of male mice. The proposed mechanism may involve the reduction of renal OATs transporter activity by E2, leading to decreased concentrations of ALI in the renal tubules. This reduction may mitigate its toxic effects on epithelial cells and diminish the production of its harmful metabolites, thereby alleviating AAI-induced nephrotoxicity.