Reactive oxygen species (ROS) generated by a variety of endogenous factors and roles in embryonic stem (ES) cells has yet to be identified. Thus, we examined role of arachidonic acid (AA) in H2O2-indued proliferation of mouse ES cells and its related signaling molecules. AA release was maximally increased in response to 10−4 M H2O2 for 1 h. In addition, H2O2 increased intracellular Ca2+ concentration ([Ca2+]i) and the phosphorylation of protein kinase C (PKC), p44/42, p38 mitogen-activated protein kinase (MAPK), and JNK/SAPK. Moreover, H2O2 induced an increase in the phosphorylation of epidermal growth factor receptor (EGFR), which was blocked by the inhibition of p44/42 or p38 MAPKs. The inhibition of each signal molecule with specific inhibitors blocked H2O2-induced cytosolic phospholipase A2 (cPLA2) activation and AA release. H2O2 increased NF-κB phosphorylation to induce an increase in the levels of cyclooxygenase (COX)-2 proteins. Subsequently, H2O2 stimulated PGE2 synthesis, which was reduced by the inhibition of NF-κB activation. Moreover, each H2O2 or PGE2 increased DNA synthesis and the number of cells. However, H2O2-induced increase in DNA synthesis was inhibited by the suppression of cPLA2 pathway. In conclusion, H2O2 increased AA release and PGE2 production by the upregulation of cPLA2 and COX-2 via Ca2+/PKC/MAPKs and EGFR transactivation, subsequently proliferation of mouse ES cells. J. Cell. Biochem. 106: 787–797, 2009. © 2009 Wiley-Liss, Inc.