MicroRNAs are reported to correlate with synaptic plasticity and exert functions in Alzheimer's disease (AD) pathogenesis. miR-369-5p is identified to be upregulated in AD mice. This study explores miR-369-5p roles in synaptic plasticity in hippocampal cells and in an AD mouse model. Wild-type C57BL/6J mice (6 months) were trained in a well-established object memory task. Two identical objects were presented to mice for 10 min. miR-369-5p expression in hippocampus, cortex, and striatum, and in hippocampal cells was measured by reverse transcription quantitative polymerase chain reaction. Then, 0.2 mM glycine and 100 nM amyloid-beta oligomers were used to treat primary hippocampal cells. The levels of plasticity-related proteins in hippocampal cells and hippocampus were evaluated by Western blotting. Object location memory (OLM) of 3xTg-AD mice was tested in an OLM protocol at 13 months of age. In this study, learning increased miR-369-5p level in the hippocampus. The increased levels of plasticity-related proteins induced by chemical long-term potentiation were inhibited by miR-369-5p inhibitors in hippocampal cultures. miR-369-5p upregulation rescued the Aβo-induced suppression in the levels of plasticity-related proteins in hippocampal cultures. miR-369-5p elevation increased GluA1 and GluA2 protein levels and rescued OLM impairment in an AD mouse model. In conclusion, miR-369-5p positively regulates the levels of plasticity-related proteins in hippocampal cultures and in an AD mice model.