Background/Objectives: Gastric cancer (GC) is one of the devastating cancers with frequent metastasis. The non-receptor tyrosine kinase Src may trigger oncogenic signaling, which is a critical target for preventing GC progression. Another potential GC target is the programmed death-1 ligand (PD-L1), which is one of the immune checkpoint molecule responsible for immunosuppression. Moreover, PD-L1 may directly influence cancer progression by regulating tumor-intrinsic events. Interestingly, Src has intimate relationship with tumor-intrinsic PD-L1 in terms of signal transduction. In this study, whether tumor-intrinsic PD-L1 interacts with Src to regulate GC progression was investigated. Methods: Western blot was used for analyzing expression of PD-L1 and activity of Src. Trans-well migration and invasion assays were for investigating whether PD-L1 and Src are required for GC progression in vitro. Lipid nanoparticles SNALPs carrying PD-L1 siRNA was for in vivo knockdown of PD-L1. Results: Experiments using Src inhibitors coupled with PD-L1 knockdown indicated PD-L1 and Src be both upstream and downstream of each other in conventional AGS and patient-derived SD110 GC cells. By scavenging reactive oxygen species (ROS) using an antioxidant dithiothreitol (DTT), both Src activity and PD-L1 expression were found to be ROS signaling-dependent. The ROS-mediated PD-L1/Src signaling was required for migration and invasion of AGS and SD110. Co-targeting Src and PD-L1 suppressed migration of AGS and SD110 more efficiently than single targeting. In the SCID mice, in vivo knockdown of PD-L1 reduced Src activity and metastasis of SD110 tumor from stomach to liver. Conclusions: co-targeting Src and tumor intrinsic PD-L1 might be a promising therapeutic approach in GC therapy.