This study investigates how tau, a microtubule-associated protein in neurons, differentially affects the motility of dynein and kinesin, two motor proteins that transport cellular cargo along microtubules. Using single-molecule studies, the researchers found that tau inhibits kinesin motility more effectively than dynein. Kinesin tends to detach from microtubules when encountering tau, while dynein often reverses direction. The microtubule-binding domain of tau is sufficient to inhibit motor activity, suggesting that tau can spatially regulate the balance of microtubule-dependent axonal transport. Tau is expressed in neurons as multiple splice forms, with different isoforms varying in the number of microtubule-binding repeats and the length of the projection domain. The study focused on tau23 and tau40, the shortest and longest isoforms, respectively. Fluorescently labeled tau proteins were used to visualize their distribution on microtubules, revealing that tau forms patches along the microtubule surface. These patches were stable over several minutes, allowing direct observation of motor interactions with tau. Kinesin-GFP motors were observed to detach from microtubules when encountering tau patches, while dynein-dynactin-GFP motors tended to reverse direction. The effect of tau on motor function was concentration- and isoform-dependent. Tau23 had a more pronounced inhibitory effect on kinesin than on dynein, while tau40 was less potent. Neither tau23 nor tau40 significantly affected motor velocity. Structurally, tau may inhibit motor function through its projection domain and/or microtubule-binding domain. Truncated versions of tau23, such as K35 and K33, were found to be stronger inhibitors than full-length tau23, indicating that the microtubule-binding domain is sufficient for motor inhibition. Differences in the net charge of the projection domain correlate with their ability to inhibit motor binding, suggesting that the acidic projection domain may mimic the acidic tail of tubulin and electrostatically recruit motor proteins to the microtubule surface. The study proposes a model in which tau controls the balance of microtubule-dependent axonal transport by locally modulating motor function. In a healthy neuron, tau is distributed in a proximal-to-distal gradient, allowing kinesin to efficiently bind to microtubules and initiate anterograde transport, while dynein can drive retrograde transport. In Alzheimer's disease, tau accumulation in the somatodendritic compartment impairs kinesin-driven anterograde transport, leading to neurodegeneration. The differential sensitivity of dynein and kinesin to tau provides a mechanism for spatiotemporal regulation of axonal transport.This study investigates how tau, a microtubule-associated protein in neurons, differentially affects the motility of dynein and kinesin, two motor proteins that transport cellular cargo along microtubules. Using single-molecule studies, the researchers found that tau inhibits kinesin motility more effectively than dynein. Kinesin tends to detach from microtubules when encountering tau, while dynein often reverses direction. The microtubule-binding domain of tau is sufficient to inhibit motor activity, suggesting that tau can spatially regulate the balance of microtubule-dependent axonal transport. Tau is expressed in neurons as multiple splice forms, with different isoforms varying in the number of microtubule-binding repeats and the length of the projection domain. The study focused on tau23 and tau40, the shortest and longest isoforms, respectively. Fluorescently labeled tau proteins were used to visualize their distribution on microtubules, revealing that tau forms patches along the microtubule surface. These patches were stable over several minutes, allowing direct observation of motor interactions with tau. Kinesin-GFP motors were observed to detach from microtubules when encountering tau patches, while dynein-dynactin-GFP motors tended to reverse direction. The effect of tau on motor function was concentration- and isoform-dependent. Tau23 had a more pronounced inhibitory effect on kinesin than on dynein, while tau40 was less potent. Neither tau23 nor tau40 significantly affected motor velocity. Structurally, tau may inhibit motor function through its projection domain and/or microtubule-binding domain. Truncated versions of tau23, such as K35 and K33, were found to be stronger inhibitors than full-length tau23, indicating that the microtubule-binding domain is sufficient for motor inhibition. Differences in the net charge of the projection domain correlate with their ability to inhibit motor binding, suggesting that the acidic projection domain may mimic the acidic tail of tubulin and electrostatically recruit motor proteins to the microtubule surface. The study proposes a model in which tau controls the balance of microtubule-dependent axonal transport by locally modulating motor function. In a healthy neuron, tau is distributed in a proximal-to-distal gradient, allowing kinesin to efficiently bind to microtubules and initiate anterograde transport, while dynein can drive retrograde transport. In Alzheimer's disease, tau accumulation in the somatodendritic compartment impairs kinesin-driven anterograde transport, leading to neurodegeneration. The differential sensitivity of dynein and kinesin to tau provides a mechanism for spatiotemporal regulation of axonal transport.