Tau is a conformationally dynamic microtubule-associated protein expressed at high levels in neurons. It is localized primarily in the axonal compartment where it has been implicated in a number of intracellular functions, including microtubule stabilization, cross-linking between the cytoskeleton and plasma membrane, acting as a scaffold for a variety of signaling molecules, and modulating the axonal transport process. Tau's myriad of functions are likely to be related to its conformationally dynamic structure, but the structure/function relationships within this molecule remain poorly defined. Additionally, Tau is known to be regulated by phosphorylation at numerous Ser/Thr and Tyr sites throughout the molecule, but the effects of phosphorylation on Tau's structural dynamics are also unclear. Previous in vitro work in our lab has shown that Tau interconverts between static and mobile (diffusive) states on the microtubule surface in an isoform and microtubule-lattice specific manner (McVicker et al., (2014) Cytoskeleton 71:184). To further extend these studies into a physiologically relevant environment in which Tau is naturally regulated by phosphorylation, we have used single-molecule imaging to examine the dynamic behavior of fluorescently-labeled Tau on the surface of microtubules within the isolated axoplasm of the squid giant axon. Tau maintains its isoform-specific ability to interconvert between static and diffusive states on the microtubule surface under these conditions. Furthermore, we demonstrate phosphorylation influences Tau's dynamic behavior on the microtubule surface, e.g., inhibition of CDK5 by Roscovitine results in a significant shift in Tau's dynamic equilibrium towards the diffusive state. These studies establish the isolated axoplasm of the squid giant axon as a novel model system for studying Tau dynamics under physiologically relevant conditions, and provide new insight into the role of phosphorylation in regulating Tau's structural dynamics on the microtubule surface.