Background: Recent epidemiologic studies have found that patients with diabetes have a higher risk of gastric cancer (GC), and the long-term use of metformin is associated with a lower risk of gastric cancer. It is believed that blocking tumor energy metabolic alterations is now emerging as a new therapeutic approach of cancer. Berberine, a natural isoquinoline alkaloid, could modulate lipid metabolism and glucose homeostasis by regulating the expression of HNF4 alpha in many metabolic diseases. Here, we investigated the effect of Berberine on GC and its possible molecular mechanism through targeting HNF4 alpha. Methods and Results: (1) AGS and SGC7901 gastric cancer cells were treated with Berberine (BBR). We found that in AGS and SGC7901 cell, BBR inhibited cell proliferation in a time- and dose-dependent manner through downregulating C-myc. BBR also induced G(0)-G(1) phase arrest with the decreased expression of cyclin D1. Moreover, BBR attenuated the migration and invasion by downregulating MMP-3. (2) The lentivirus infection was used to silence the expression of HNF4 alpha in SGC7901 cell. The results demonstrated that the knockdown of HNF4 alpha in SGC7901 slowed cells proliferation, induced S phase arrest and dramatically attenuated gastric cancer cells' metastasis and invasion. (3) We performed GC cells perturbation experiments through B16015 (an HNF4 alpha antagonist), AICAR (an AMPK activator), Compound C (AMPK-kinase inhibitor), metformin and BBR. Our findings indicated that BBR downregulated HNF4 alpha while upregulating p-AMPK. Moreover, the inhibition of HNF4 alpha by BBR was AMPK dependent. (4) Then the LV-HNF4 alpha-RNAi SGC7901 cell model was used to detect the downstream of HNF4 alpha in vitro. The results showed that the knockdown of HNF4 alpha significantly decreased WNT5A and cytoplasmic beta-catenin, but increased E-cadherin in vitro. Berberine also downregulated WNT5A and cytoplasmic beta-catenin, the same as LV-HNF4 alpha-RNAi and B16015 in GC cells. (5) Finally, the SGC7901 and LV-HNF4 alpha-RNAi SGC7901 mouse-xenograft model to evaluate the effect of BBR and HNF4 alpha gene on GC tumor growth. The result illustrated that BBR and knockdown of HNF4 alpha suppressed tumor growth in vivo, and BBR decreased HNF4 alpha, WNT5A and cytoplasmic beta-catenin levels, the same effect as HNF4 alpha knockout in vivo. Conclusion: BBR not only had proliferation inhibition effect, attenuated the invasion and migration on GC cell lines, but also suppressed the GC tumor growth in vivo. The antigastric cancer mechanism of BBR might be involved in AMPK-HNF4 alpha-WNT5A signaling pathway. HNF4 alpha antagonists, such as BBR, could be a promising anti-gastric cancer treatment supplement.