The entorhinal cortex (EC) is regarded as the gateway to the hippocampus; the superficial layers (layers I-III) of the EC convey the cortical input projections to the hippocampus, whereas deep layers of the EC relay hippocampal output projections back to the superficial layers of the EC or to other cortical regions. The superficial layers of the EC receive strong serotonergic projections from the raphe nuclei. However, the function of serotonin in the EC is still elusive. In the present study, we examined the molecular and cellular mechanisms underlying serotonin-mediated inhibition of the neuronal excitability in the superficial layers (layers II and III) of the EC. Application of serotonin inhibited the excitability of stellate and pyramidal neurons in the superficial layers of the EC by activating the TWIK-1 type of the two-pore domain K+ channels. The effects of 5-HT were mediated via 5-HT 1A receptors and required the function of Gαi3 subunit and protein kinase A. Serotonin-mediated inhibition of EC activity resulted in an inhibition of hippocampal function. Our study provides a cellular mechanism that might at least partially explain the roles of serotonin in many physiological functions and neurological diseases.