AbstractShort-term memory deficits have been associated with prefrontal cortex (PFC) dysfunction in Alzheimer’s disease (AD) and AD mouse models. Extratelencephalic projection (ET) neurons in the PFC play a key role in short-term working memory, but the mechanism between ET neuronal dysfunction in the PFC and short-term memory impairment in AD is not well understood. Here, using fiber photometry and optogenetics, we found reduced neural activity in the ET neurons in the medial prefrontal cortex (mPFC) of the 5×FAD mouse model led to object recognition memory (ORM) deficits. Activation of ET neurons in the mPFC of 5×FAD mice rescued ORM impairment, and inhibition of ET neurons in the mPFC of wild type mice impaired ORM expression. ET neurons in the mPFC that project to supramammillary nucleus were necessary for ORM expression. Viral tracing and in vivo recording revealed that mPFC ET neurons received fewer cholinergic inputs from the basal forebrain in 5×FAD mice. Furthermore, activation of cholinergic fibers in the mPFC rescued ORM deficits in 5×FAD mice, while acetylcholine deficiency reduced the response of ET neurons in the mPFC to familiar objects. Taken together, our results revealed a neural mechanism behind ORM impairment in 5×FAD mice.