Lansoprazole, a proton pump inhibitor, can exert antioxidant effects through the induction of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, independently of the inhibition of acid secretion in the gastrointestinal tract. Lansoprazole has been reported to provide hepatoprotection in a drug-induced hepatitis animal model through the Nrf2/heme oxygenase-1 (HO1) pathway. We sought to investigate the molecular mechanism of cytoprotection by lansoprazole. Anin vitroexperimental model was conducted using cultured rat hepatic cells treated with lansoprazole to analyze the expression levels of Nrf2 and its downstream genes, the activity of Nrf2 using luciferase reporter assays, cisplatin-induced cytotoxicity, and signaling pathways involved in Nrf2 activation. Lansoprazole treatment of rat liver epithelial RL34 cells induced transactivation of Nrf2 and the expression of the Nrf2-dependent antioxidant genes encoding HO1, NAD(P)H quinone oxidoreductase-1, and glutathione S-transferase A2. Furthermore, cycloheximide chase experiments revealed that lansoprazole prolongs the half-life of the Nrf2 protein. Notably, cell viability was significantly increased by lansoprazole treatment in a cisplatin-induced cytotoxicity model. Moreover, the siRNA knockdown of Nrf2 fully abolished the cytoprotective effect of lansoprazole, whereas the inhibition of HO1 by tin-mesoporphyrin only partially abolished this. Finally, lansoprazole promoted the phosphorylation of p38 mitogen-activated protein kinase (MAPK) but not that of the extracellular signal-regulated kinase or the c-Jun N-terminal kinase. Using SB203580, a specific inhibitor for p38 MAPK, the lansoprazole-induced Nrf2/antioxidant response elements pathway activation and cytoprotective effects were shown to be exclusively p38 MAPK dependent. Lansoprazole was shown by these results to exert a cytoprotective effect on liver epithelial cells against the cisplatin-induced cytotoxicity through the p38 MAPK signaling pathway. This could have potential applications for the prevention and treatment of oxidative injury in the liver.