Ca²⁺/calmodulin-dependent protein kinase (CaMK) is an important downstream target of Ca²⁺ in the hypertrophic signaling pathways. We previously showed that the activation of apoptosis signal-regulating kinase 1 (ASK1) or NF-κB is sufficient for cardiomyocyte hypertrophy. Infection of isolated neonatal cardiomyocytes with an adenoviral vector expressing CaMKIIδ3 (AdCaMKIIδ3) induced the activation of ASK1, while KN93, an inhibitor of CaMKII, inhibited phenylephrine-induced ASK1 activation. Overexpression of CaMKIIδ3 induced characteristic features of in vitro cardiomyocyte hypertrophy. Infection of cardiomyocytes with an adenoviral vector expressing a dominant negative mutant of ASK1 (AdASK(KM)) inhibited the CaMKIIδ3-induced hypertrophic responses. Overexpression of CaMKIIδ3 increased the κB-dependent promoter/luciferase activity and induced IκBα degradation. Coinfection with AdCaMKIIδ3 and AdASK(KM), and pre-incubation with KN93 attenuated CaMKIIδ3- and phenylephrine-induced NF-κB activation, respectively. Expression of a degradation resistant mutant of IκBα inhibited CaMKIIδ3-induced hypertrophic responses. These results indicate that CaMKIIδ3 induces cardiomyocyte hypertrophy mediated through ASK1-NF-κB signal transduction pathway.