Cyclooxygenase-2 (COX-2) and COX-2-induced prostaglandin E2 (PGE2) have been implicated in all stages of malignant tumorigenesis. Although many aspects of matrix metalloproteinase (MMP2) on tumor invasion have been studied, the exact mechanism of PGE2-induced MMP2 overproduction has not been clearly defined. We have previously demonstrated that PGE2-enhanced extracellular signal-regulated kinase (Erk) phosphorylation via EP1 signaling pathway involved in PGE2-induced cell proliferation. Based on the identification of the transcription factor cyclic AMP response element-binding protein (CREB) as an important regulator of MMP2 and Erk phosphorylate CREB at ser133, we hypothesize that CREB may be implicated in the signaling of PGE2 stimulation to MMP2 overproduction via EP1 receptor. In the study, we investigated the role of EP1 receptor on PGE2-induced MMP2 expression and delineated the signaling pathway that contributes to EP1 receptor modulation of MMP2 in human cholangiocarcinoma cells. We found PGE2 or selective EP1 receptor agonist 17-P-T-PGE2-stimulated MMP2 expression and selective EP1 receptor antagonist SC-51322 or EP1 receptor siRNA abrogated PGE2-induced MMP2 expression. Intracellular calcium chelator BAPTA-AM, the selective inhibitor of EGFR AG1478 and the selective inhibitor of Erk PD98059 blocked EP1 receptor activation-induced CREB phosphorylation and MMP2 expression. A novel dominant-negative (D-N) inhibitor protein of the CREB, termed A-CREB, attenuated EP1 receptor activation-induced MMP2 expression. Our findings suggest that PGE2-enhanced MMP2 expression is, at least in part, mediated through EP1 receptors and calcium signaling pathway-induced CREB phosphorylation in human cholangiocarcinoma cells.