Background <p>Gastric cancer (GC), a malignant neoplasm that arises from the epithelium of the gastric mucosa, endangers patients’ lives and health severely. Regulator of G-protein signaling 16 (RGS16) has been found to be correlated with the malignant progression of various cancers, and BHLHE40 is highly expressed in GC. However, it remains unclear whether there is a regulatory mechanism between the them.</p> Methods <p>The bioinformatics tools were applied to assess the differentially expressed genes in GC. Next, the expression levels of mRNA and protein were evaluated by qRT-PCR and Western blot. Cellular behaviors were assessed using CCK-8, EdU, Transwell, and flow cytometry assays. Meanwhile, the ferroptosis-related indicators were measured. Subsequently, the xenograft models were set up to estimate the role of RGS16 in vivo. Besides, the interaction between BHLHE40 and RGS16 was determined using ChIP assay and dual-luciferase reporter assay.</p> Results <p>RGS16 exhibited an upregulated pattern in GC. In addition, silencing RGS16 impeded the proliferation, migration and invasion of GC cells while reinforcing apoptosis and ferroptosis. Moreover, RGS16 boosted the growth of tumors in vivo. Furthermore, BHLHE40 could bind to RGS16 and positively regulate its expression. Overexpression of RGS16 reversed the effects of silencing BHLHE40 on GC cells.</p> Conclusion <p>BHLHE40 curbed ferroptosis and oxidative stress of GC cells by modulating the expression of RGS16, thereby facilitating the malignant progression of GC.</p>