The neuronal-voltage gated sodium channel (VGSC), Na_V1.6, plays an important role in propagating action potentials along myelinated axons. Calmodulin (CaM) is known to modulate the inactivation kinetics of Na_V1.6 by interacting with its IQ motif. Here we report the crystal structure of apo-CaM:Na_V1.6IQ motif, along with functional studies. The IQ motif of Na_V1.6 adopts an α-helical conformation in its interaction with the C-lobe of CaM. CaM uses different residues to interact with Na_V1.6IQ motif depending on the presence or absence of Ca²⁺. Three residues from Na_V1.6, Arg1902, Tyr1904 and Arg1905 were identified as the key common interacting residues in both the presence and absence of Ca²⁺. Substitution of Arg1902 and Tyr1904 with alanine showed a reduced rate of Na_V1.6 inactivation in electrophysiological experiments in vivo. Compared with other CaM:Na_V complexes, our results reveal a different mode of interaction for CaM:Na_V1.6 and provides structural insight into the isoform-specific modulation of VGSCs.