Abstract Sonic hedgehog signaling pathway is an important regulator during the initiation and progression of pancreatic cancer, and sustained activation of this pathway contributes to excessive deposition of tumor stroma and maintenance of tumor-initiating cells. Cyclopamine (CPA) is a potent inhibitor of the SHH pathway. In this work, we encapsulated both paclitaxel (PTX) and CPA in a long-circulating polymeric micelles to obtain M-CPA/PTX. In a genetically engineered mouse model of pancreatic ductal adenocarcinoma (PDAC) (KPC model), systemic administration of M-CPA/PTX significantly prolonged the median survival of mice with well-established tumors from 23 days with nab-paclitaxel or gemcitabine to 58 days post-enrollment (p<0.0001). Further studies indicated that M-CPA/PTX modulated PDAC stroma by softening extracellular matrix, increasing blood perfusion, and alleviating hypoxia without depletion of α-smooth muscle actin-positive tumor-associated fibroblasts. Significantly, combination of M-CPA/PTX and anti-PD-1 immunotherapy significantly prolonged the survival of PDAC-bearing KPC mice compare with monotherapies (p<0.01). In an immune-competent, orthotopic PDAC model bearing established murine PDAC, treatments with combined M-CPA/PTX and anti-PD-1 enhanced tumor infiltration of CD8+ T cells and IFNγ expression. Neutralization of either CD8 T cells or INFγ nullified the therapeutic gain achieved with combination therapy. Thus, M-CPA/PTX enhanced checkpoint blockade immunotherapy through modulation of PDAC stroma and enhanced tumor infiltration of effector immune cells. Citation Format: Jun Zhao, Zhilan Xiao, Willem Overwijk, Chun Li. Pharmacological modulation of PDAC stroma to enhance checkpoint blockade immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1014.