Differential expression, activity, phosphorylation, and oligomerization of tau play a critical role during neuronal development and in a number of age-related neurodegenerative diseases. An experimental system that accurately models the molecular changes involved in tauopathies, particularly changes in tau isoform activity, requires the expression at physiological levels of the full complement of tau isoforms present in the adult human brain. To this end, we analyzed tau expression in human cortical neurons (HCNs) in culture. Here, we show that the isoform profile of tau in HCNs is similar to that in the adult human brain and that isoform expression is regulated during neuronal development and by cellular substrates. Interestingly, 4R tau exhibited a distinct pattern of expression and subcellular localization, suggesting the presence of specific functional roles for tau isoforms in HCNs. Tau phosphorylation, microtubule binding, and subcellular localization were markedly altered by pharmacological manipulation of tau-directed phosphatase activities, which also induced the appearance of tau oligomeric forms associated with memory loss in animal models of tauopathy. Thus, experimentally induced changes in tau activity and function in HCNs recapitulate critical features of tauopathies that may lead to neuronal dysfunction and degeneration in the human brain.