Pancreatic endocrine cells (EC) are responsible for glucose homeostasis through the coordination of glucagon and insulin from alpha and beta cells, respectively. For the treatment of diabetes, in vitro differentiation of pancreatic ECs from human pluripotent stem cells is massively studied. During the pancreatic EC development, endocrine progenitor cells (EP) transiently express NEUROGENIN 3 (NGN3). NGN3 is a basic helix-loop-helix transcription factor that is activating EC-associated genes such as NEUROD1, PAX6, PAX4, and NKX2.2. It lasts only a few hours in individual EPs, however, its deficiency leads to pancreatic EC agenesis. Therefore, understanding the NGN3 regulation mechanism in EPs is critical for EC differentiation. It is known that multi-phosphorylation of NGN3 by CDKs is important for its transcriptional activity and ubiquitin-mediated degradation, but other modifications are not elucidated yet. Arginine methylation of non-histone proteins regulates protein functionality and stability. PRMT1 is a predominant type I arginine methyl-transferase in mammalian cells. In this study, a PRMT1-inducible knockout (KO, P-iKO) hESC line was generated to discover the role of PRMT1 during pancreatic EC differentiation in vitro. PRMT1 depletion in EPs leads to accumulation of NGN3 and impaired EC development. Using the NGN3-T2A-eGFP hESC line, it was confirmed that PRMT1-KO does not alter NGN3 transcripts itself. Based on these results, Arginine 65 methylation of NGN3 by PRMT1 is essential for proper EC development from hESCs. Also, this modification was critical for transcriptional activity and rapid ubiquitin-mediated degradation of NGN3.