Calcium/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional serine/threonine protein kinase that transmits calcium signals in various cellular processes. CaMKII is activated by calcium-bound calmodulin (Ca/CaM) through a direct binding mechanism involving a regulatory C-terminal α-helix in CaMKII. The Ca/CaM binding triggers transphosphorylation of critical threonine residues proximal to the CaM-binding site leading to the autoactivated state of CaMKII. The demonstration of its critical roles in pathophysiological processes has elevated CaMKII to a key target in the management of numerous diseases. The molecule KN-93 is the most widely used inhibitor for studying the cellular and in vivo functions of CaMKII. It is widely believed that KN-93 binds directly to CaMKII， thus preventing kinase activation by competing with Ca/CaM. Herein， we employed surface plasmon resonance， NMR， and isothermal titration calorimetry to characterize this presumed interaction. Our results revealed that KN-93 binds directly to Ca/CaM and not to CaMKII. This binding would disrupt the ability of Ca/CaM to interact with CaMKII， effectively inhibiting CaMKII activation. Our findings also indicated that KN-93 can specifically compete with a CaMKIIδ-derived peptide for binding to Ca/CaM. As indicated by the surface plasmon resonance and isothermal titration calorimetry data， apparently at least two KN-93 molecules can bind to Ca/CaM. Our findings provide new insight into how in vitro and in vivo data obtained with KN-93 should be interpreted. They further suggest that other Ca/CaM-dependent， non-CaMKII activities should be considered in KN-93-based mechanism-of-action studies and drug discovery efforts.