The ciliary rootlet is a large striated fibrous network originating from basal bodies in ciliated cells. To explore its postulated role in intracellular transport, we investigated the interaction between kinesin light chains (KLCs) and rootletin, the structural component of ciliary rootlets. We show here that KLCs directly interact with rootletin and are located along ciliary rootlets. Their interactions are mediated by the heptad repeats of KLCs. Further studies found that these interactions tethered kinesin heavy chains along ciliary rootlets. However, the ciliary rootlet-bound kinesin-1 did not recruit microtubules or move along ciliary rootlets. Additionally, amyloid precursor protein (APP; a kinesin-1 vesicular cargo receptor) and presenilin 1 (a presumed cargo of APP/kinesin-1) were found to be enriched along the rootletin fibers, suggesting that the interaction between ciliary rootlets and kinesin-1 recruits APP and presenilin 1 along ciliary rootlets. These findings indicate that ciliary rootlets may provide a scaffold for kinesin-1 vesicular cargos and, thus, play a role in the intracellular transport in ciliated cells.