Abstract Pancreatic ductal adenocarcinoma (PDAC) is the deadliest cancer due lack of early diagnosis and inherent refractory to traditional chemo- and even novel immune-therapies. PDAC is marked by its extensive desmoplastic stroma containing extracellular matrix (ECM), vasculature and most importantly cancer-associated fibroblasts (CAFs). CAFs that exist in heterogenous subtypes are responsible for shaping PDAC tumor microenvironment being the main source of: ECM deposition leading up to desmoplasia that hampers the efficacy of various therapeutics, as well as diverse cytokines and growth factors that promote the growth of cancer cells and the shift towards dedifferentiated and invasive phenotype and contribute towards immune suppressive microenvironment. Thus, modulating CAFs phenotype is an attractive mean to increase the efficacy of therapies targeting PDAC cells. Here we assessed the potential therapeutic benefits of Isorhamnetin, a flavonoid previously shown to exert anti-proliferative, anti-fibrotic and anti-inflammatory effect in various cancer types, to modulates PDAC microenvironment by characterizing Isorhamnetin effect on human PDAC-derived CAFs. Isorhamnetin inhibited CAFs proliferation, induced apoptosis, and cell cycle arrest by impairing mitochondrial health. Importantly, isorhamnetin attenuates the expression of inflammatory CAFs (iCAFs) signature including IL1A, IL6, LIF, CXCL1 and promotes phenotype shift towards myofibroblasts (myCAFs)-as evident in higher level of αSMA positive CAFs. Our results demonstrate antiproliferative effect of isorhamnetin on human PDAC-derived CAFs and a phenotype switch from iCAFs to myCAFs and warrant further investigation for its use in combinatorial therapy that target both the cancer and the tumor microenvironment as a whole. Citation Format: Munkhzul Ganbold, Pakavarin Louphrasitthiphol, Yoshihiro Miyazaki, Tatsuya Oda, Kenichi Tominaga, Hiroko Isoda. Isorhamnetin restricts cancer-associated fibroblasts (CAFs) phenotypic plasticity, poising them towards tumor-restraining myCAFs: Potential use of isorhamnetin as neoadjuvant in pancreatic ductal adenocarcinoma (PDAC) [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C068.