Growing evidence suggests that dysregulated microRNAs were critical in the development of tumors and the progression number of malignancies. This research aimed to check the effect of microRNA 320a-3p transfection on gastric cancer (GC) cell lines. Following transfection, the efficacy was determined by the RT-PCR method. After that, MTT, scratch assay, DAPI staining, RT-PCR, and flow cytometry were used respectively. The results demonstrated that the viability of GC cells considerably decreased following transfection. Moreover, microRNA 320a-3p transfection significantly suppressed cell migration and induced apoptosis in these cells. We found that transfection of microRNA 320a-3p remarkably decreased PD-L1 gene expression and influenced epithelial-mesenchymal transition (EMT)-related and apoptotic gene expressions. The findings propose that microRNA 320a-3p could decrease cell proliferation and migration and induce apoptosis by increasing TP53 and CASP3 expression levels in GC cells. Notably, microRNA 320a-3p might be a potential target in GC immunotherapy by suppressing the PD-L1 gene expression.