Abstract PIN1 is a phosphorylation-directed prolyl isomerase that alters the conformation and, therefore, the function of many proteins. Due to its role in activation and stabilization of many oncogenes, we hypothesized that targeting PIN1 in pancreatic ductal adenocarcinoma (PDA) would slow tumor growth. We tested this hypothesis in vitro and in vivo with PIN1 inhibitors and/or genetic model systems. Pancreatic cancer cell lines knocked down for PIN1 or treated with PIN1 inhibitors showed decreased proliferation, invasion, and anchorage independent growth compared to control lines. Consistent with these in vitro results, treatment of pancreatic cancer xenografts or genetically engineered p48-Cre; LSL-KrasG12D; p53R172H (KPC) mice with PIN1 inhibitors decreased tumor growth and extended overall survival. Similar results were seen in KPC mice that were crossed into a full body PIN1 knockout (PIN1−/−). Further analysis of KPC PIN1−/− tumors revealed not only reduced size of pancreatic tumors, but also decreased alpha-SMA expression and decreased ECM deposition in the stroma surrounding the tumors. PDA is characterized by a dense, desmoplastic tumor stroma that contributes to tumor growth, metastasis, and therapeutic resistance. Pancreatic stellate cells (PSCs) that are activated in the tumor microenvironment play a major role in the deposition of ECM and secrete growth factors to support tumor cell proliferation and survival. To interrogate a direct role for PIN1 in the stroma, we first orthotopically injected a KPC cell line into syngeneic PIN1+/+ or PIN1−/− mice and found dramatic reduction of tumor cell growth in PIN1−/− hosts. Next, we analyzed PSCs in vitro and found that loss of PIN1 reduces their proliferation and alters their secretion of paracrine factors that support oncogenic phenotypes. For example, PSCs with loss of PIN1 have reduced expression of HGF and increased expression of SPINT1 and SPINT2, inhibitors of HGF activation. Conditioned media from control PSCs, but not from PSCs lacking PIN1 expression, activates the MET receptor on cancer cell lines, resulting in altered cancer cell phenotypes. In addition, we show that loss of PIN1 in PSCs inhibits TGF-beta induced stellate cells activation into a myofibroblast phenotype. Single cell ATAC-seq analysis demonstrated that a subset of TGF-beta responsive chromatin changes are impaired in the absence of PIN1. Our ongoing work utilizes 2D co-cultures, heterotypic 3D bioprinted tissues, and in vivo mouse models to interrogate the mechanisms by which fibroblast phenotypes and the tumor-stromal crosstalk is impacted by PIN1. Citation Format: Ellen M. Langer, Isabel A. English, Kayleigh M. Kresse, Kevin MacPherson, Brittany L. Allen-Petersen, Colin J. Daniel, Andrew Adey, Rosalie C. Sears. The prolyl isomerase PIN1 plays a critical role in fibroblast plasticity to impact pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT012.