This study aimed to investigate the pivotal role of cystatin B (CSTB) in the development of gastric cancer and to explore its possible regulatory mechanism. Human gastric cancer SGC-7901 cells as a model in vitro were transfected with plasmid PCDNA3.1-CSTB and siRNA-CSTB using Lipofectamine 2000. Quantitative real-time PCR (qRT-PCR) and Western blotting were performed to determine the relative expression of CSTB and PI3K/Akt/mTOR pathway-related protein. Moreover, MTT assay, Transwell assay, and flow cytometry were used to assess cell proliferation, migration, and apoptosis, respectively. The results showed that CSTB was significantly downregulated in SGC-7901 cells compared with gastric epithelial cells. CSTB was successfully overexpressed and suppressed after cells were transfected with pc-CSTB and si-CSTB, respectively. Moreover, cell viability and migration were significantly decreased after being transfected with pc-CSTB when compared with the control group, while being obviously increased after transfection with si-CSTB. However, cell apoptosis was significantly induced after being transfected with pc-CSTB, while being obviously suppressed after transfection with si-CSTB. Besides, the expression levels of p-PI3K, p-Akt, and p-mTOR proteins were all significantly decreased in the pc-CSTB transfection group when compared with the control group, while being increased in the si-CSTB transfection group. Our findings suggest that CSTB downregulation may promote the development of gastric cancer by affecting cell proliferation and migration, and the PI3K/Akt/mTOR signaling pathway was activated in this process. CSTB may serve as a potential therapeutic target for gastric cancer.