Immunotherapy is one of the most promising strategies for cancer, compared with traditional treatments. As one of the key emerging immunotherapies, anti-PD-1/PD-L1 treatment has brought survival benefits to many advanced cancer patients. However, in pancreatic cancer, immunotherapy-based approaches have not achieved a favorable clinical effect because of mismatch repair deficiencies. Therefore, the majority of pancreatic tumors are regarded as immune-quiescent tumors and non-responsive to single-checkpoint blockade therapies. Many preclinical and clinical studies suggest that it is still important to clarify the regulatory mechanism of the PD-1/ PD-L1 pathway in pancreatic cancer. As a marker of cancer stem cells, DCLK1 has been found to play an important role in the occurrence and development of a plethora of human cancers. Recent researches have revealed that DCLK1 is closely related to EMT process of tumor cells, meanwhile, it could also be used as a biomarker in gastrointestinal tumors to predict the prognoses of patients. However, the role that DCLK1 plays in the immune regulation of tumor microenvironments remains unknown. Therefore, we sought to understand if DCLK1 could positively regulate the expression of PD-L1 in pancreatic cancer cells. Furthermore, we examined if DCLK1 highly correlated with the Hippo pathway through TCGA database analysis. We found that DCLK1 helped regulate the level of PD-L1 expression by affecting the corresponding expression level of yes-associated protein in the Hippo pathway. Collectively, our study identifies DCLK1 as an important regulator of PD-L1 expression in pancreatic tumor and highlights a central role of DCLK1 in the regulation of tumor immunity.