This study investigates the molecular mechanisms underlying gefitinib resistance in lung adenocarcinoma (LUAD) cells harboring EGFR mutations. The authors found that gefitinib's inhibitory effect on EGFR-mutated LUAD cells was partially dependent on the induction of ferroptosis, and ferroptosis protection led to gefitinib resistance. Among the ferroptosis suppressors, aldo-keto reductase family 1 member C1 (AKR1C1) showed significant upregulation in gefitinib-resistant LUAD cells and predicted poor progression-free survival (PFS) and overall survival (OS) in patients treated with first-generation EGFR-TKIs. Knockdown of AKR1C1 partially reversed drug resistance by sensitizing LUAD cells to gefitinib-mediated ferroptosis. The decreased expression of miR-338-3p contributed to the upregulation of AKR1C1 in gefitinib-resistant LUAD cells. Furthermore, long non-coding RNA (lncRNA) NEAT1_1 sponged miR-338-3p to neutralize its suppression on AKR1C1. Dual-luciferase reporter assays and miRNA rescue experiments confirmed the NEAT1_1/miR-338-3p/AKR1C1 axis in EGFR-mutated LUAD cells. Gain- and loss-of-function assays demonstrated that the NEAT1_1/miR-338-3p/AKR1C1 axis promoted gefitinib resistance, proliferation, migration, and invasion in LUAD cells. This study reveals the role of the NEAT1_1/miR-338-3p/AKR1C1 axis in gefitinib resistance in LUAD and suggests that targeting this axis might be a novel strategy to overcome gefitinib resistance in LUAD harboring EGFR mutations.This study investigates the molecular mechanisms underlying gefitinib resistance in lung adenocarcinoma (LUAD) cells harboring EGFR mutations. The authors found that gefitinib's inhibitory effect on EGFR-mutated LUAD cells was partially dependent on the induction of ferroptosis, and ferroptosis protection led to gefitinib resistance. Among the ferroptosis suppressors, aldo-keto reductase family 1 member C1 (AKR1C1) showed significant upregulation in gefitinib-resistant LUAD cells and predicted poor progression-free survival (PFS) and overall survival (OS) in patients treated with first-generation EGFR-TKIs. Knockdown of AKR1C1 partially reversed drug resistance by sensitizing LUAD cells to gefitinib-mediated ferroptosis. The decreased expression of miR-338-3p contributed to the upregulation of AKR1C1 in gefitinib-resistant LUAD cells. Furthermore, long non-coding RNA (lncRNA) NEAT1_1 sponged miR-338-3p to neutralize its suppression on AKR1C1. Dual-luciferase reporter assays and miRNA rescue experiments confirmed the NEAT1_1/miR-338-3p/AKR1C1 axis in EGFR-mutated LUAD cells. Gain- and loss-of-function assays demonstrated that the NEAT1_1/miR-338-3p/AKR1C1 axis promoted gefitinib resistance, proliferation, migration, and invasion in LUAD cells. This study reveals the role of the NEAT1_1/miR-338-3p/AKR1C1 axis in gefitinib resistance in LUAD and suggests that targeting this axis might be a novel strategy to overcome gefitinib resistance in LUAD harboring EGFR mutations.