Pancreatic ductal adenocarcinoma (PDAC) is one of the most common malignant tumors of the pancreas. Preclinical studies show that it evades the immune system with immune checkpoints and promotes tumor development. V-domain Ig suppressor of T cell activation (VISTA) is a new immune-check point from the B7 family and is highly expressed in cancer cells. Overexpression of toll like receptor 4 (TLR4) in pancreatic adenocarcinoma is associated with induced tumorigenesis, tumor growth, resistancy to chemotherapy. Naloxone is an opioid and inhibits TLR4-ligand association. In this study, we investigated the relation of TLR4 and downstream pathways with immune-check point VISTA in pancreatic cancer proliferation. We initially collected pancreatic cancer-related datasets using the GEPIA2 and UALCAN databases. Based on this data obtained the effect of various concentrations and incubation times of naloxone were used on PANC-1 cells proliferation. A combination of naloxone and VISTA-siRNA were applied, and the effect of both naloxone and combined treatment on TLR4, Interleukin 1 receptor associated kinase 4 (IRAK4) and VISTA gene expression were analyzed in pancreatic cancer cells. As a result of analysis with Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR), gene expression levels of TLR4, IRAK4 and VISTA were significantly suppressed and cell proliferation was significantly reduced. We found that administration of naloxone and VISTA-siRNA in combination with PDAC cells suppressed signaling. Therefore, we considered that the relationship between VISTA and TLR4 signaling pathways and the other possible associated signal molecules may be an important marker in determining the response of immune checkpoint inhibitors in cancer treatment.