The activity of Ca(2+)/calmodulin-dependent protein kinase IV (CaMKIV) is sensitive to activity-dependent changes in the level of intracellular Ca(2+). Following neuronal stimulation, the activation of CaMKIV may trigger synaptic modifications and transcriptional responses, both of which are involved in regulating cognitive and emotional behavior. Here, we used CaMKIV knockout (KO) neurons and mice to examine the function of CaMKIV in Ca(2+)-stimulated intracellular signaling and animal behavior, respectively. Following NMDA receptor activation or membrane depolarization, the up-regulation of CREB (cAMP responsive element binding protein) and its target gene Bdnf (brain-derived neurotrophic factor) was intact in cortical neurons obtained from CaMKIV KO mice. CaMKIV KO mice displayed severe impairment in contextual fear memory but normal locomotor activity and anxiety level in the contextual training chamber. Although CaMKIV KO mice showed normal memory in the standard passive avoidance task, they were defective in learning the temporal dissociative passive avoidance task. As indicated by the light/dark test and marble-burying test data, CaMKIV KO mice showed less anxiety and normal perseveration. In the voluntary wheel-running test, CaMKIV KO mice showed normal running time and distance but higher maximal running speed. Our results demonstrate the function of CaMKIV in regulating different forms of fear memory, anxiety, and certain aspect of motor function.