Cantharidin (CTD), extracted from the traditional Chinese medicine mylabris, has shown significant curative effects against a variety of tumors, but its clinical application is limited by its nephrotoxicity, and the underlying molecular mechanisms remain unclear. In this study, we investigated the toxic effects in mouse kidneys following CTD treatment by pathological observations, biochemical index detection, and transcriptomics, and explored the underlying molecular mechanisms by RNA sequencing (RNA-seq). The results showed that after CTD exposure, the kidneys had different degrees of pathological damage, altered uric acid and creatinine levels in serum, and the antioxidant indexes in tissues were significantly increased. RNA-seq analysis revealed 674 differentially expressed genes compared with the control group, of which 131 were upregulated and 543 were downregulated. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses showed that many differentially expressed genes were closely related to the stress response, the CIDE protein family, and the transporter superfamily, as well as the MAPK, AMPK, and HIF-1 pathways. The reliability of the RNA-seq results was verified by qRT-PCR. These findings offer new insight into the molecular mechanisms of renal toxicity caused by CTD.