Deficient signaling by insulin, as occurs in diabetes, is associated with impaired brain function, and diabetes is associated with an increased prevalence of Alzheimer's disease. One of the hallmark pathological characteristics of Alzheimer's disease is the presence of neurofibrillary tangles containing hyperphosphorylated tau, a microtubule-associated protein. Therefore, we tested the hypothesis that insulin depletion caused by administration of streptozotocin may cause tau hyperphosphorylation in mouse brain by using site-specific phosphorylation-dependent tau antibodies to obtain precise identification of the phosphorylation of tau on individual residues. A massive (fivefold average increase) and widespread at multiple residues (detected with eight different phosphorylation-dependent tau antibodies) increase in the phosphorylation of tau was found in mouse cerebral cortex and hippocampus within 3 days of insulin depletion by streptozotocin treatment. This hyperphosphorylation of tau at some sites was rapidly reversible by peripheral insulin administration. Examination of several kinases that phosphorylate tau indicated that they were unlikely to account for the widespread hyperphosphorylation of tau caused by streptozotocin treatment, but there was a large decrease in mouse brain protein phosphatase 2A activity, which is known to mediate tau phosphorylation. These results show that insulin deficiency causes rapid and large increases in tau phosphorylation, a condition that could prime tau for the neuropathology of Alzheimer's disease, thereby contributing to the increased susceptibility to Alzheimer's disease caused by diabetes.