Calcineurin (CN) is a heterodimer of a catalytic subunit, calcineurin A (CNA), and a regulatory subunit (CNB). Here, we find that the mechanism by which CNB regulates CNA depends on the substrate involved. The regulation mechanism involving tau and its truncation segments is distinct from that involving RII peptide, and the efficiencies of CNA to dephosphorylate tau are constant regardless of whether CNB was present or not. The findings shed some light on the role of CNB in controlling phosphorylation of tau in vivo and the pathogenesis of tauopathies such as Alzheimer's Disease.