Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family that binds to and activates the EGF receptor, and is expressed in a variety of tissues, lung, heart, brain and skeletal muscle. HB-EGF is transactivated by angiotensin II, ET-1 and various growth factors in cardiomyocyte and is known to induce cardiac hypertrophy via PI3-Akt, MAP kinase, JAK-STAT pathway. However, relatively less is known pertaining to ERK5 pathway in HB-EGF induced cardiac hypertrophy. In the present study, we tried to elucidate that MEK5-ERK5 pathway is involved in HB-EGF induced cardiomyocyte hypertrophy. HB-EGF (10 ng/ml) significantly induced [3H]-leucine incorporation and fetal gene atrial natriuretic factor (ANF) mRNA expression in H9c2 cells. In addition, HB-EGF induced activation of ERK5 pathway and pretreatment with EGFR inhibitor, AG1478 attenuated the activation of ERK5. MEK5, upstream regulator of ERK5, inhibition using siRNA attenuated cardiomyocyte hypertrophy suggesting involvement of EGFR-ERK5 pathway in HB-EGF induced cardiac hypertrophy. We further analyzed on cyclooxygenase-2 (COX-2), one of downstream molecule ERK5, involving in hypertrophic signaling. In our study, HB-EGF induced expression of COX-2 (1 ~ 4 hour) in H9c2 cells and rofecoxib, COX-2 inhibitor inhibited HB-EGF-induced ANF mRNA expression. Furthermore, HB-EGF-induced COX-2 expression was attenuated by MEK5 siRNA transfection in H9c2 cells. In conclusion, HB-EGF induces cardiomyocyte hypertrophy through EGFR-ERK5-COX-2 pathway. Our findings will help us better understand the molecular mechanism of HB-EGF induced cardiomyocyte hypertrophy.