COX-2-derived PGE2 has been implicated in the development of various types of cancers. However, the exact mechanism of PGE2-induced cancer cell proliferation and survival is still unclear. In the current study, the mechanism underlying PGE2-enhanced Erk phosphorylation in human cholangiocarcinoma cells was determined. The intracellular concentration of calcium in three cholangiocarcinoma cell lines was measured using a laser confocal scanning microscope and the expression levels of Erk and EGFR phosphorylation were determined by Western blot analyses. The activation of EP1 receptors involved in PGE2-stimulated Erk activation and increasing the intracellular concentration of calcium was elucidated using selective EP1 receptor subtype antagonists and agonist. The intracellular calcium chelator, BAPTA-AM, was shown to block PGE2-induced Erk and EGFR phosphorylation. PGE2-induced Erk phosphorylation was abrogated by pretreatment with the EGF receptor kinase inhibitor, AG1478. Our findings suggest that in human cholangiocarcinoma cells, PGE2-enhanced phosphorylation of Erk is, at least in part, mediated through EP1 receptors and EGFR phosphorylation induced by increases in the intracellular concentration of calcium.