Assemblies of tau can transit between neurons, seeding aggregation in a prion-like manner. To accomplish this, tau must cross cell-limiting membranes, a process that is poorly understood. Here, we establish assays for the study of tau entry into the cytosol as a phenomenon distinct from uptake, in real time, and at physiological concentrations. The entry pathway of tau is cell type specific and, in neurons, highly sensitive to cholesterol. Depletion of the cholesterol transporter Niemann-Pick type C1 or extraction of membrane cholesterol renders neurons highly permissive to tau entry and potentiates seeding even at low levels of exogenous tau assemblies. Conversely, cholesterol supplementation reduces entry and almost completely blocks seeded aggregation. Our findings establish entry as a rate-limiting step to seeded aggregation and demonstrate that dysregulated cholesterol, a feature of several neurodegenerative diseases, potentiates tau aggregation by promoting entry of tau assemblies into the cell interior.