Alzheimer’s disease (AD), the most prevalent neurodegenerative disease, is characterized in part by disruptions in axonal transport. Axonal transport is a process by which motor proteins carry organelles and other cargo made in the neuronal cell body along microtubule tracks to distal regions of the axon. The microtubule-associated protein (MAP) Tau plays a crucial role in regulating axonal transport, and is implicated in the development of AD and other types of dementia collectively known as Tauopathies. Tau is a neuronal-specific MAP that has six isoforms alternatively spliced from a single gene. These isoforms differ by the presence of zero, one, or two N-terminal acidic inserts and three or four C-terminal microtubule binding repeats. Tau is also known to be an intrinsically disordered protein that undergoes a dynamic equilibrium between static and diffusive states on the microtubule surface. The dynamics of Tau are important in the regulation of motor protein mediated axonal transport in neurons. Isoform-specific differences in the dynamic behavior of Tau on the microtubule surface, however, are not yet fully understood. Diffusive Tau is thought to be stabilized by electrostatic interactions between its N- and C-termini while static Tau is proposed to be extended with its C-terminal repeats contacting the microtubule and the N-terminus projected away from the microtubule surface. Thus, the N-terminal inserts may help regulate Tau’s dynamic behavior and function during axonal transport. In this study, the dynamics of two different isoforms of Tau, both with three-microtubule binding repeats but a different number of N-terminal acidic inserts, were assessed using single molecule imaging techniques and novel data analysis methods.