AIM: To investigate the inhibitory effect and mechanism of hesperetin (HST) on human gefitinib-resistant NCI-H1975 lung adenocarcinoma cells. METHODS: CCK-8 assay was used to detect the effects of HST on the proliferation of NCI-H1975 cells; Annexin V-FITC/PI double staining was used to detect HST-induced apoptosis of NCI-H1975 cells; flow cytometry was used to observe the effects of HST and HST + acetylcysteine (NAC) combined on the levels of reactive oxygen species (ROS) in NCI-H1975 cells; Western blot was used to detect the expression of Bcl-2, Bax, Cleaved Caspase-3, p-eIF2α, eIF2α and CHOP proteins in NCI-H1975 cells by HST, NAC + HST and Salubrinal + HST. The antitumor effect of HST in vivo was studied by constructing a xenograft model in nude mice; HE staining was used to observe the effect of HST on the histopathological morphology of heart, liver, kidney and xenograft in tumor-bearing mice; immunohistochemistry was used to detect the effect of HST on p-eIF2α protein in xenograft tissues. RESULTS: Compared with the control group, HST at 37.5 μmol/L for 24 h significantly inhibited the viability of NCI-H1975 cells (P<0.05), and NAC attenuated the inhibitory effect of HST; concentrations greater than 150 μmol/L increased intracellular ROS levels (P<0.05), induced apoptosis (P<0.05), and increased Caspase3 activity (P<0.01), and compared with HST 300 μmol/L group, ROS levels, apoptosis rate, and Caspase3 activity were significantly decreased in NAC + HST 300 μmol/L group (P<0.01); HST up-regulated Bax, Cleaved Caspase-3, CHOP, and p-eIF2α expression and down-regulated Bcl-2 expression in a concentration-dependent manner (P<0.01), and compared with HST 300 μmol/L group, Bax and Cleaved Caspase-3 expression was decreased and Bcl-2 expression was increased in Sal + HST 300 μmol/L group; p-eIF2α and CHOP expression were significantly down-regulated in the NAC + HST 300 μmol/L and Sal + HST 300 μmol/L groups (P<0.01). In vivo, experiments showed that HST could significantly inhibit the growth of transplanted tumors and up-regulate p-eIF2α protein expression (P<0.05), and had no significant adverse effects on the growth status, body weight and important organs (heart, liver and kidney) of nude mice. CONCLUSION: HST inhibits the proliferation of gefitinib-resistant NCI-H1975 lung adenocarcinoma cells in vitro and in vivo, and the mechanism may be related to HST mediating ER stress-induced apoptosis of NCI-H1975 cells through ROS.