Background and Purpose Epiberberine (EPI), one of the active ingredients in Coptidis Rhizoma, exhibited excellent potential for inhibiting gastric cancer (GC) in our previous study. Nevertheless, the underlying mechanism was still unclear. The aim of this study was to identify the core receptor target of EPI in GC and to explore the underlying mechanisms. Experimental Approach The identification and validation of receptor targets were performed using multiple bioinformation methods and in vitro experiments. The co-localization and binding sites between EPI and GABRB3 were detected by confocal microscope, molecular dynamic, point mutation and cellular thermal shift assay (CETSA). The inhibition effects and mechanisms of EPI on the malignant phenotypes of GC cells via GABRB3 signaling were determined by MTT, colony formation, wound closure assay, flow cytometry, real-time qPCR, Western blot analysis, chloride and calcium ion fluorescent probes, dual-luciferase assay and a tumor-bearing mouse model. Key Results Initial studies discovered that EPI might stimulate the GABAA receptor-composed GABAergic system with GABRB3 as the core in GC cells. EPI might directly bind to the heteropentamer via residue Tyr157, Phe200, and Thr202. EPI overactivating GABRB3 made an abnormal accumulation of intracellular Ca2+ and subsequently induced p21/CDK1/CCNB1 axis-dependent G2/M cell cycle arrest. EPI binding also promoted the accumulation of endonuclear p-CREB1 via the Ca2+/PKC-δ/CaMKII pathway, which formed a positive feedback loop that upregulated GABRB3 expression as well as promoted p53 expression. Conclusion and Implications This study may prolong the understanding for the anti-GC mechanism of EPI and provide a new strategy for GC treatment.