Hyperphosphorylation of the microtubule associated protein tau (tau) is inextricably linked to several neurodegenerative diseases, collectively termed tauopathies, in which synapse dysfunction occurs through largely unidentified mechanisms. Our research aimed to uncover molecular mechanisms by which phosphorylation of tau (pTau) affects synapse function. Using combined molecular and electrophysiological analysis with in vitro genetic knock-in of phosphorylation mutant human tau in male rat CA1 hippocampal neurons, we show an interplay between tau and protein kinase C and casein kinase substrate in neurons protein 1 (PACSIN1) that regulates synapse function. pTau at serine residues 396/404 decreases tau:PACSIN1 binding and evokes PACSIN1-dependent functional and structural synapse weakening. Knockdown of tau or PACSIN1 increases AMPA receptor-mediated current at extrasynaptic regions, supporting a role for these proteins in affecting AMPA receptor trafficking. The pTau-induced PACSIN1 dissociation may represent a pathophysiological regulator of synapse function that underlies tauopathy-associated synapse defects.Significance StatementKnowledge is still lacking for how hyperphosphorylation of tau and its effectors lead to synaptic and neuronal dysfunction. Our results provide crucial insight for this mechanistic understanding; we show that specific tau phosphorylation events modulate its protein interaction with PACSIN1 and thus elicits synapse weakening likely through PACSIN1-dependent regulation of AMPA receptor trafficking. These findings develop our understanding of molecular events that may be relevant to cellular changes underpinning tauopathy-associated neurodegenerative diseases.