Defective axonal transport has been proposed as an underlying mechanism that may give rise to neurodegeneration. We investigated the effect of phosphorylation on the axonal transport of tau, a neuronal protein that stabilizes microtubules and is hyperphosphorylated and mislocalized in Alzheimer's disease. We report here that specific inhibition of glycogen synthase kinase-3 (GSK-3) reduces tau phosphorylation and significantly decreases the overall rate of axonal transport of tau in rat cortical neurons. Tau mutants, with serine/threonine targets of GSK-3 mutated to glutamate to mimic a permanent state of phosphorylation, were transported at a significantly increased rate compared to wild-type tau. Conversely, tau mutants, in which alanine replaced serine/threonine to mimic permanent dephosphorylation, were transported at a decreased rate compared to wild-type tau. We also found that tau interacts with the light chain of kinesin-1 and that this is dependent on the phosphorylation state of tau. Tau phosphorylation by GSK-3 increased binding, and dephosphorylated tau exhibited a reduced association with kinesin-1. We conclude that GSK-3 phosphorylation of tau modulates its axonal transport by regulating binding to kinesin-1. Hyperphosphorylated tau in Alzheimer's disease appearing first in distal portions of axons may result from aberrant axonal transport of phosphorylated tau reported here.