Mitochondria are mobile organelles and cells regulate mitochondrial movement in order to meet the changing energy needs of each cellular region. Ca²⁺ signaling, which halts both anterograde and retrograde mitochondrial motion, serves as one regulatory input. Anterograde mitochondrial movement is generated by kinesin-1, which interacts with the mitochondrial protein Miro through an adaptor protein, milton. We show that kinesin is present on all axonal mitochondria, including those that are stationary or moving retrograde. We also show that the EF-hand motifs of Miro mediate Ca²⁺-dependent arrest of mitochondria and elucidate the regulatory mechanism. Rather than dissociating kinesin-1 from mitochondria, Ca²⁺-binding permits Miro to interact directly with the motor domain of kinesin-1, preventing motor/microtubule interactions. Thus, kinesin-1 switches from an active state in which it is bound to Miro only via milton, to an inactive state in which direct binding to Miro prevents its interaction with microtubules. Disrupting Ca²⁺-dependent regulation diminishes neuronal resistance to excitotoxicity.