Human embryonic stem cells (hESCs) possess the ability to differentiate into various cell types, which is intricately linked to fatty acid synthesis and metabolism. Fatty acid desaturase 2 (FADS2) plays important role in fatty acid metabolism. In this study, we elucidate that the inhibition of FADS2 by SC-26196 enhances hESC pluripotency by upregulating key pluripotency genes such as POU5F1, NANOG, and KLF5. Moreover, SC-26196 treatment alters the fatty acid metabolic profile of hESCs, decreasing the synthesis of saturated fatty acids (SFAs) while increasing the content of monounsaturated fatty acids (MUFAs). Meanwhile, transcriptomic and proteomic analyses revealed that under FADS2 inhibition, hESCs maintain pluripotency primarily through enhanced oxidative phosphorylation and modified fatty acid metabolism. Knockdown and overexpression experiments confirm that FADS2 is a crucial regulator of these metabolic processes, and is essential for sustaining hESCs pluripotency. Collectively, this study unveils the pivotal role of FADS2 in the metabolic regulation of hESCs and provide new insights into the mechanisms governing pluripotency.