The roles of calmodulin (CaM) have been key points of controversy in the regulation of inositol-1,4,5-trisphosphate receptor (IP(3)R). To address the issue, we studied the interaction between CaM and the suppressor domain of IP(3)R, a key allosteric regulatory domain. First, by means of a pulldown and a fluorescence titration experiment, we confirmed the interaction. Through subsequent NMR binding experiments, we observed dramatic peak disappearances of the suppressor domain on interaction with apo-CaM. The data indicated that apo-CaM induces large-scale dynamic conformational changes in the suppressor domain, involving partial unfolding and subdomain rearrangement. Analysis of the NMR data of CaM surprisingly revealed that its C lobe alone can cause such changes. Further binding experiments showed that calcium allows the free N lobe to bind to the suppressor domain, which induces extra conformational changes in both of the proteins. These results were also confirmed with CaM deletion mutants with either the N or C lobe. On the basis of this novel binding mechanism, we propose a model in which the partial unfolding of the suppressor domain by apo-CaM and the stepwise binding of the N lobe of CaM to the suppressor domain are important elements of calcium/CaM inhibition of IP(3)R. We believe that our working model encompasses previous regulation mechanisms of IP(3)R by calcium/CaM and provides new insights into the CaM-target interaction.