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BACKGROUND Lead (Pb) is a highly toxic and persistent substance that easily accumulates in living organisms, eliciting cellular toxicity and oxidative stress. Some selenium-containing proteins and peptides prepared from plant extracts are beneficial for protecting the body's health and resisting external disturbances. In this study, selenium-containing peptide species were prepared from selenium-enriched Pleurotus eryngii protein hydrolysates and to evaluate the benefits of selenium-containing peptides on Pb-induced oxidative stress in NCTC1469 hepatocytes. RESULTS Trypsin was selected as primary enzyme to hydrolyze the selenium-enriched protein (SPH). The optimal hydrolysis conditions were: hydrolysis time, 1.5 h; initial pH, 8.0. The SPH was digested by trypsin and then purified by ultrafiltration, gel filtration chromatography and RP-HPLC to obtain the selenium-containing peptides SPH-I-2. Furthermore, SPH-I-2 was analyzed and a number of total 12 selenium-containing peptides were identified by LC-MS/MS. The NCTC1469 cell culture study showed that selenium-containing peptides were capable of reducing ROS levels and regulating the Keap1/Nrf2 pathway by upregulating Nrf2, HO-1, GCLC, GCLM, NQO1 genes and downregulating Keap1 genes. Moreover, selenium-containing peptides were also able to suppress Pb-induced elevated levels of NO, LDH and MDA, increase antioxidant enzyme activity and alleviate cell apoptosis. CONCLUSION The present research indicated that the selenium-containing peptides could protect cells from Pb2+ -induced oxidative stress. This article is protected by copyright. All rights reserved.