Background <p>Ferroptosis is a novel regulatory mechanism of cell death caused by iron-dependent lipid peroxidation (lipid-ROS) accumulation. The aim of this study was to explore whether dihydrolipoic acid transacetylase (DLAT) influences ferroptosis to promote the progression of gastric cancer (GC).</p> Methods <p>The expression of DLAT in GC tissues and cell lines was examined by qRT-PCR, western blotting, and immunohistochemistry. Kaplan–Meier survival curve analysis was used to estimate overall survival, and the log-rank test was used to estimate recurrence-free survival. The functional role of DLAT in GC was evaluated using in vitro and in vivo experiments including MTT, scratch healing tests, transwell assays and a xenograft tumor mouse model. Ferroptosis was evaluated by using malondialdehyde (MDA), reduced glutathione (GSH), lactate dehydrogenase (LDH), and superoxide dismutase (SOD) detection kits, iron accumulation assays, lipid ROS quantification, and mitochondrial deep red fluorescence staining kits. Western blotting was used to detect the biomarkers of ferroptosis and the interactions between DLAT and Nrf2, HO-1 and GPX4.</p> Results <p>We found that DLAT was upregulated in GC cells and tissues, and high DLAT expression indicated poor prognosis. Knockdown of DLAT inhibited the proliferation, invasion and metastasis of GC cells by increasing the levels of MDA and LDH, promoting the accumulation of Fe²⁺ and lipid ROS, consuming GSH and SOD, reducing mitochondrial membrane potential, and suppressing the expression of GPX4. Overexpression of GPX4 can specifically salvage the growth inhibition of GC cells caused by DLAT knockdown, but did not affect the activity of the upstream DLAT, Nrf2 and HO-1. The inhibition of ferroptosis by DLAT was related to the regulation of the Nrf2/HO-1/GPX4 antioxidant axis. In <i>vivo</i>, DLAT inhibited the Nrf2/HO-1 axis, hindering the growth of transplanted tumors.</p> Conclusion <p>These results indicated that DLAT deficiency increased accumulation of Fe²⁺ and lipid ROS, enhanced disruption of mitochondrial membrane potential, and induced ferroptosis through the Nrf2/HO-1/GPX4 axis, thereby inhibiting the malignant progression of GC cells. DLAT serves as a prognostic biomarker and potential therapeutic target for GC.</p> Trial registration <p>Registry and the Registration of the study/trial: N/A.</p>