Background and objective: Mammalian sterile 20-like kinase 1 (Mst1) plays a critical role in regulating cell survival and apoptosis. However, its influence on gastric cancer cell viability is not understood. Our study aims to explore the specific role of Mst1 in gastric cancer. Materials and methods: Cellular viability was measured via TUNEL staining, MTT assays, and Western blotting. Immunofluorescence was performed to observe mitochondrial fission. Mst1 overexpression assays were conducted to observe the regulatory mechanisms of Mst1 in mitochondrial fission and cell apoptosis. Results: The results demonstrated that Mst1 was downregulated in AGS cells when compared with GES-1 cells. However, overexpression of Mst1 reduced cell viability and increased apoptosis in AGS cells. Molecular experiments showed that Mst1 overexpression mediated mitochondrial damage, as evidenced by decreased ATP production, increased ROS generation, more cyt-c translocation from the mitochondria into the cytoplasm and nucleus, and activated the caspase-9-related apoptotic pathway. Furthermore, we found that mitochondrial fission was required for Mst1-induced mitochondrial dysfunction; inhibition of mitochondrial fission sustained mitochondrial homeostasis in response to Mst1 overexpression. In addition, our data revealed that Mst1 controlled mitochondrial fission via repressing the AMPK-Sirt3 pathway. Activation of the AMPK-Sirt3 pathway negated the promoting effect of Mst1 overexpression on mitochondrial fission. Conclusion: Altogether, our data identified Mst1 as a novel tumor-suppressive factor in promoting cell death in gastric cancer cells by triggering mitochondrial fission and blocking the AMPK-Sirt3 axis.