It was recently found that glutamine (Gln) supplementation activates glutamatergic neurotransmission and prevents chronic-stress-induced mild cognitive impairment (MCI). In this study, we evaluated the effects of Gln on glutamatergic activity in the medial prefrontal cortex and the onset of cognitive impairment in a triple-transgenic Alzheimer’s disease mouse model (3×Tg-AD). Female 3×Tg-AD mice were fed a normal diet (3×Tg) or a Gln-supplemented diet (3×Tg+Gln) from 2 to 6 months of age. Glutamatergic neuronal activity was analyzed at 6 months, and cognitive function was examined at 2, 4, and 6 months. 3×Tg mice exhibited a decrease in glutamatergic neurotransmission in the infralimbic cortex, but 3×Tg+Gln mice did not. The 3×Tg group showed MCI at 6 months of age, but the 3×Tg+Gln group did not. The expressions of amyloid peptide, inducible nitric oxide synthase, and IBA-1 were not elevated in the infralimbic cortex in the 3×Tg+Gln group. Therefore, a Gln-supplemented diet could delay the onset of MCI even in a mouse model predisposed to cognitive impairment and dementia through genetic modification.