Piper nigrum Linnaeus is often used as a treatment for chills, stomach diseases, and other ailments. Piperine has many biological functions; however, its mechanism for preventing gastric mucosal damage is still unclear. The objective of this study was to investigate the preventive effects of piperine on ethanol-induced gastric mucosal injury by using GES-1 cells and rats. SOD, CAT, GSH-Px and MDA were effectively regulated in GES-1 cells pre-treated with piperine. Piperine significantly increased SOD, CAT and GSH-Px activities, but decreased the ulcer area, MDA, ROS and MPO levels in the gastric tissues of rats. RT-PCR analysis showed that piperine downregulated the mRNA expression levels of keap1, JNK, ERK and p38, and upregulated the mRNA transcription levels of Nrf2 and HO-1. Western blotting results indicated that piperine could activate the protein expression levels of Nrf2 and HO-1 and inhibit the protein expression levels of keap1, p-JNK, p-ERK and p-p38. In conclusion, piperine suppressed ethanol-induced gastric ulcers in vitro and in vivo via oxidation inhibition and improving gastric-protecting activity by regulating the Nrf2/HO-1 and MAPK signalling pathways.