The microtubule associated protein tau is a major component of neurofibrillary tangles in Alzheimer disease brain, however the neuropathological processes behind the formation of neurofibrillary tangles are still unclear. Previously, 14‐3‐3 proteins were reported to bind with tau. 14‐3‐3 Proteins usually bind their targets through specific serine/threonine –phosphorylated motifs. Therefore, the interaction of tau with 14‐3‐3 mediated by phosphorylation was investigated. In this study, we show that the phosphorylation of tau by either protein kinase A (PKA) or protein kinase B (PKB) enhances the binding of tau with 14‐3‐3 in vitro. The affinity between tau and 14‐3‐3 is increased 12‐ to 14‐fold by phosphorylation as determined by real time surface plasmon resonance studies. Mutational analyses revealed that Ser214 is critical for the phosphorylation‐mediated interaction of tau with 14‐3‐3. Finally, in vitro aggregation assays demonstrated that phosphorylation by PKA/PKB inhibits the formation of aggregates/filaments of tau induced by 14‐3‐3. As the phosphorylation at Ser214 is up‐regulated in fetal brain, tau’s interaction with 14‐3‐3 may have a significant role in the organization of the microtubule cytoskeleton in development. Also as the phosphorylation at Ser214 is up‐regulated in Alzheimer’s disease brain, tau’s interaction with 14‐3‐3 might be involved in the pathology of this disease.