Tau hyperphosphorylation is a major neuropathological hallmark of many neurodegenerative disorders such as Alzheimer's disease. Several anesthetics have been shown previously to induced marked tau hyperphosphorylation. Although the ketamine/xylazine mixture is one of the most commonly used anesthetic agents in animal research and veterinary practice, the effect of this anesthetic agent on tau phosphorylation still remains to be determined. Here, we found that ketamine-/xylazine-induced a rapid and robust hyperphosphorylation of tau in a dose-dependent manner under normothermic and hypothermic conditions in mice. When used together, ketamine and xylazine exerted a synergistic action on tau phosphorylation most strongly not only on epitopes S396 and S262, but also on other residues (T181, and S202/T205). We observed that activation of the calmodulin-dependent protein kinase II (CaMKII) is the major upstream molecular event leading to tau hyperphosphorylation following ketamine/xylazine anesthesia in mice. Moreover, we observed that intracerebroventricular injection of the selective CaMKII inhibitor KN93 attenuated tau hyperphosphorylation. Since ketamine/xylazine also had a marked impact on other key molecular signaling pathways involving the MAP/microtubule affinity-regulating kinase (MARK), extracellular signal-regulated kinase (ERK), and glycogen synthase kinase-3 (GSK3), our study calls for high caution and careful monitoring when using this anesthetic agent in laboratory animal settings across all fields of biological sciences in order to avoid artifactual results.