Neuroinflammation and disruptions in glutamatergic neurotransmission are hallmark features of Alzheimer’s disease (AD). Various compounds have been investigated for their potential to modulate these processes in this disease. Tacrolimus (FK506), a calcineurin inhibitor (CNI), has been suggested as a candidate for the treatment of AD, although its effects and possible mechanisms have not been extensively evaluated. Here we investigated whether tacrolimus treatment could mitigate cognitive deficits, neurotoxicity, and microgliosis in AD models, including Aβ_1–42-induced intrahippocampal damage and middle-aged transgenic APP/PS1 mice, as well as improve glutamate release dysregulation in synaptosomes from the latter. A single dose of tacrolimus prevented the cognitive impairment induced by intrahippocampal microinjection of Aβ_1–42 in the novel object recognition test (NORT), and reduced the neurodegeneration. Interestingly, in the APP/PS1 model, a 30-day treatment with the drug did not prevent memory impairment in the NORT, albeit it improved the social interaction and partially reduced microgliosis. Finally, tacrolimus restored the intrasynaptotosomal calcium levels and normalized impaired glutamate release in synaptosomes from APP/PS1 mice. These findings provide new evidence that both acute and chronic treatment with tacrolimus exerts neuroprotective effects, providing a foundation for the potential therapeutic application of this CNI in managing AD.