Tacrolimus (TAC) has emerged as a potential therapy for Alzheimer’s disease (AD), with the challenge of balancing its therapeutic benefits against its immunosuppressive effects. This study explores the efficacy of a sub-immunosuppressive TAC dosing regimen to ameliorate AD-related pathologies. TAC was administered daily for 14 days, with drug concentrations measured via liquid chromatography tandem mass spectrometry (LC-MS/MS) in whole blood and hippocampal tissue from C57BL6J mice, while immunofluorescence analyses and Western blotting (performed on hippocampal extracts) were conducted in 10–12 month old 3xTg-AD mice to evaluate levels of tau and amyloid-beta (Aβ) proteins. The results from LC-MS/MS revealed that lower TAC doses resulted in sub-immunosuppressive blood levels, while still penetrating the hippocampi. Immunofluorescence showed reductions in tau and Aβ proteins in 3xTg-AD mice. Additionally, Western blot analyses revealed reductions in tau and Aβ, along with increases in synaptic and autophagy-related proteins. These findings highlight the potential of sub-immunosuppressive TAC doses in effectively targeting AD pathology while minimizing the risk of chronic systemic immunosuppression. Further research and clinical trials are warranted to establish the optimal TAC dosing regimen for AD treatment.