As a classic tumor suppressor pathway, Hippo signaling axis is activated by various extra-pathway factors to regulate cell differentiation and organ development. However, recent studies have reported that the activation of Hippo signaling pathway may be more dependent on the autophosphorylation of its core kinase cassette. Here, we demonstrate that protein arginine methyltransferase 5 (PRMT5) is involved in inducing the inactivation of Hippo signaling pathway in pancreatic cancer. Our study shows that the initiator serine/threonine kinase 3 (STK3, also known as MST2) of Hippo signaling pathway can be symmetrically di-methylated at arginine-461 (R461) and arginine-467 (R467) in the SARAH domain by PRMT5, and the methylated MST2 suppresses its autophosphorylation and kinase activity by blocking the formation of homodimer, thereby inactivating Hippo signaling pathway in pancreatic cancer. Moreover, we also discover that the specific PRMT5 inhibitor GSK3326595 re-activates the dysregulated Hippo signaling pathway