Tau, a member of the MAP2/tau family of microtubule-associated proteins, functions to stabilize and organize axonal microtubules in healthy neurons. In contrast, tau dissociates from microtubules and forms neurotoxic extracellular aggregates in neurodegenerative tauopathies. MAP2/tau family proteins are characterized by three to five conserved, intrinsically disordered repeat regions that mediate electrostatic interactions with the microtubule surface. We use molecular dynamics, microtubule-binding experiments and live cell microscopy to show that highly conserved histidine residues near the C terminus of each MT-binding repeat are pH sensors that can modulate tau-MT interaction strength within the physiological intracellular pH range. At lower pH, these histidines are positively charged and form cation-π interactions with phenylalanine residues in a hydrophobic cleft between adjacent tubulin dimers. At higher pH, tau deprotonation decreases microtubule-binding both in vitro and in cells.However, electrostatic and hydrophobic characteristics of histidine are required for tau-MT-binding as substitution with constitutively positively charged, nonaromatic lysine or uncharged alanine greatly reduces or abolishes tau-MT binding. Consistent with these findings, tau-MT binding is reduced in a cancer cell model with increased intracellular pH but is rapidly rescued by decreasing pH to normal levels. Thus, these data add a new dimension to the intracellular regulation of tau activity and could be relevant in normal and pathological conditions.Neuronal development and function relies on a class of structural microtubule (MT)-associated proteins (MAPs) that share conserved MT-binding motifs. These MAP2/tau family proteins associate with MTs in an extended conformation along the MT protofilament ridge (1, 2), and binding is thought to be mediated largely through electrostatic interactions of basic amino acids distributed throughout the MAP2/tau family MTbinding repeats with the negatively charged MT wall. Tau (MAPT), the most intensely studied member of the MAP2/tau family, is expressed in pH-modulated tau-microtubule binding