Background: Aucubin is pharmacologically active natural compound which possesses numerous beneficial properties in vitro and in vivo. Purpose: This study aimed to evaluate the protective effect of aucubin against cisplatin (CP)-induced acute kidney injury in mice and the mechanism of its action. In addition, a modulation of CP-induced cyototoxicity was investigated in vitro. Study design: Aucubin was administrated to mice orally (po) and intraperitoneally (ip) (1.5 and 5 mg/kg) for two consecutive days, two days after ip injection of cisplatin (CP), 11 mg/kg. Human cervical cancer HeLa cells were treated with CP 30 µM and aucubin 0–1000 μM. Methods: Hematoxylin and eosin staining, western blot, immunohistochemistry and immunofluorescence were used to evaluate the renoprotective activity of aucubin. The cell viability assay was used to evaluate in vitro cytotoxicity of aucubin. Results: Treatment with aucubin by both routes of administration ameliorated histopathological changes and reduced elevated serum markers of kidney injury. CP administration increased renal expression of HO-1 and 4-HNE, as well as TNF-α, which was dose-dependently ameliorated by the administration of aucubin, indicating the suppression of oxidative stress and inflammation, respectively. Moreover, aucubin reduced increased renal expression of cleaved caspase-3 and -9 and decreased PARP cleavage, suggesting amelioration of CP-induced apoptosis. Mechanistically, aucubin normalized the activation of ERK1/2, PI3K/Akt, FOXO3a, STAT3 and NF-κB signaling in mice kidneys. Aucubin was slightly more successful in the amelioration of kidney injury when administered by ip than by po route. In human cervical cancer cells, aucubin did not modulate the CP cytotoxicity in doses up to 1 mM. Conclusions: The findings of this study show that aucubin acts as a protective agent against CP-induced nephrotoxicity, without interference with the CP anticancer activity in clinically relevant doses.