General anesthesia increases the risk for cognitive impairment and Alzheimer's disease (AD) in vulnerable individuals such as the elderly. We previously reported that prior administration of insulin through intranasal delivery can prevent the anesthesia-induced cognitive impairment and biochemical changes in the brain. However, little is known about the underlying molecular mechanisms. Here, we report that general anesthesia resulted in downregulation of mammalian/mechanistic target of rapamycin (mTOR) and eukaryotic elongation factor 2 (eEF2) in the brain along with reduction of presynaptic proteins and brain-derived neurotrophic factor and cognitive impairment in aged mice. Prior administration of intranasal insulin prevented these anesthesia-induced changes. These results suggest the involvement of the mTOR-eEF2 signaling pathway in the anesthesia-induced brain changes and cognitive impairment and in the prevention of these changes with insulin. Correlation analyses and the use of eEF2 kinase inhibitor further support our conclusions. These studies shed light on the molecular mechanism by which anesthesia and insulin could act on synaptic proteins and cognitive function.