We have recently reported that nicotine enhances object recognition memory (ORM) via acting on the medial prefrontal cortex (mPFC) in mice. However, it remains unclear whether this effect of nicotine is mediated by the activation of mPFC neurons. Additionally, since the mPFC sends excitatory afferents to the perirhinal cortex (PRH), it is hypothesized that this pathway is involved in the nicotine-induced enhancement of ORM. To address these issues, we performed the novel object recognition test (NOR) using adeno-associated virus (AAV) vector-mediated chemogenetic techniques in male C57BL/6J mice (7 – 16 weeks old). Systemic nicotine (0.1 mg/kg; s.c.) administration before the training session of NOR significantly increased ORM. Silencing of mPFC neurons with inhibitory DREADD, which were selectively expressed in excitatory neurons, before nicotine injection significantly inhibited the nicotine-induced ORM enhancement. Conversely, activation of mPFC neurons with excitatory DREADD before the training session enhanced ORM. Moreover, selective silencing of PRH-projecting mPFC neurons with inhibitory DREADD before nicotine administration also attenuated the nicotine-induced ORM enhancement. These data suggest that nicotine enhances ORM via activating mPFC neurons, which may then activate the PRH.