Gastric cancer is a leading cause of mortality worldwide. Alpha, 2′-dihydroxy-4,4′-dimethoxydihydrochalcone is a type of limonoid mainly isolated from Cedrela odorata (Meliaceae) that has been shown to suppress cell proliferation in several human carcinoma cell lines. In this study, we investigated the anti-cancer ability of alpha, 2′-dihydroxy-4,4′-dimethoxydihydrochalcone and its underlying mechanism in MKN45 cells. Alpha, 2′-dihydroxy-4,4′-dimethoxydihydrochalcone induced excess reactive oxygen species (ROS) accumulation. Transwell and wound healing assays demonstrated that alpha, 2′-dihydroxy-4,4′-dimethoxydihydrochalcone inhibited the invasion and migration ability of MKN45 cells. Moreover, autophagy-related proteins Beclin-1, Atg5, and Atg7 were up-regulated. Light chain 3 (LC3)-I protein was converted into LC3-II under alpha, 2′-dihydroxy-4,4′-dimethoxydihydrochalcone exposure. Transmission electron microscopy demonstrated that alpha, 2′-dihydroxy-4,4′-dimethoxydihydrochalcone treatment resulted in the formation of autophagosomes. Immunofluorescence assays suggested that alpha, 2′-dihydroxy-4,4′-dimethoxydihydrochalcone treatment elicited dot formation of green fluorescent protein (GFP)-LC3. 3-methyladenine (3-MA), an autophagy inhibitor, demonstrated that autophagy promoted death in MKN45 cells. Western blotting showed that ROS/mitogen activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways play crucial roles in the intrinsic mechanism of alpha, 2′-dihydroxy-4,4′-dimethoxydihydrochalcone’s activity. The combined use of N-acetyl-_L-cysteine (NAC) or U0126 validated the regulatory role of ROS/MEK/ERK signaling pathways. Alpha, 2′-dihydroxy-4,4′-dimethoxydihydrochalcone administration inhibited the growth of MKN45 xenograft tumors in nude mice and suppressed Ki67 expression. More importantly, a similar effect was achieved in a patient-derived xenograft (PDX) model, which is more relevant to clinical application. Taken together, alpha, 2′-dihydroxy-4,4′-dimethoxydihydrochalcone has the potential to be further developed into an anti-tumor agent for clinical treatment of gastric cancer.