A novel N-acetyltransferase, Shati/Nat8l, was identified in the brains of mice exposed to methamphetamine. Shati/Nat8l overexpression in the medial prefrontal cortex (mPFC) was found to attenuate methamphetamine-induced dependence. The mPFC is a brain region that plays an important role in cognitive function. However, the effect of Shati/Nat8l on cognition and memory has not yet been clarified. To understand the role of Shati/Nat8l in memory, we generated C57BL/6J mice with overexpressed Shati/Nat8l in the mPFC and performed memory-related experiments, including novel object-location and object-in-context tests. Furthermore, we used quantitative immunohistochemistry to assess the presynaptic and postsynaptic proteins, synaptophysin and postsynaptic density protein (PSD)-95, respectively. Shati/Nat8l overexpression in the mPFC impaired both novel object-location and object-in-context memory. Moreover, Shati/Nat8l overexpression in the mPFC reduced PSD-95 levels, but not synaptophysin levels in the mPFC. These results demonstrated that Shati/Nat8l overexpression in the mPFC is involved in location and contextual memory, and can affect the excitatory postsynaptic protein, PSD-95.