Alzheimer’s disease (AD) is the most common form of dementia in the elderly, and most AD patients show defective insulin signaling. Tau protein levels are elevated in the AD brains, and the insulin signaling is impaired in many AD patients, but the direct link between tau and insulin signaling is still missing. Here, we investigated whether increasing tau affects insulin signaling. We found that overexpression of full-length human tau in neuroblastoma Neuro2a cells decreased the constitutive activity and insulin-induced activation of insulin receptor, PDK and Akt. Overexpression of tau reduced GSK-3β phosphorylation and decreased insulin-stimulated translocation of GLUT4 from an intracellular compartment to the plasma membrane. We also demonstrated that tau overexpression increased JNK phosphorylation with upregulation of the inhibitory phosphorylation of IRS-1 at serine residues and downregulation of the tyrosine phosphorylation. Furthermore, simultaneous inhibition of JNK reversed tau overexpression-induced insulin signaling impairment. These findings suggest that intracellular accumulation of tau protein inhibits insulin signaling through activation of JNK, which provides new insights in the toxicities of tau protein in AD and tauopathies.