Significance Despite its importance for a host of cellular processes and contribution to neurological, viral, and bacterial disease, the molecular mechanisms underlying the regulation of the heterotetrameric motor kinesin-1 by its light chains and the binding of its cargo are not well understood. Here, we describe how a previously unnoticed intramolecular interaction between the light chain tetratricopeptide repeat domain (KLC2 ^TPR ) and a highly conserved peptide motif within an unstructured region of the molecule occludes a key cargo binding site on the light-chain TPR domain. Cargo binding displaces this intramolecular interaction, effecting a global overall conformational change in KLCs that results in a more extended conformation. We propose a model describing how, via this molecular switch, cargo binding regulates the activity of the holoenzyme.