An acute application of neurosteroid pregnenolone sulfate (PREGS) to hippocampal slices from adult rats induced a long-lasting potentiation (LLP_PREGS) at the perforant path-granule cell synapse. As a partial mechanism of the LLP_PREGS, we previously revealed that PREGS transiently increases the probability of presynaptic glutamate release via a sensitization of 7-nicotinic acetylcholine receptor (7nAChR). We herein demonstrate that the LLP_PREGS could be separated into two independent processes: the above mentioned early presynaptic-origin short-term potentiation (STP_PREGS) and a delayed postsynaptic NMDA receptor (NMDAr)-dependent long-term potentiation termed LTP_PREGS. This study focused on the analysis of the signaling mechanism underlying the LTP_PREGS. PREGS increased the tyrosine phosphorylation of NR2B, a subunit of NMDAr, and the NMDAr-mediated Ca²⁺ influx in the granule cells. The enhanced Ca²⁺ influx was largely attenuated by the NR2B subunit inhibitor Ifenprodil and the Src kinase family inhibitor PP2. PREGS also triggered a persistent phosphorylation of extracellular signal-regulated kinase 2 (ERK2) followed by an ERK-dependent phosphorylation of cAMP-response element-binding protein (CREB), which was crucial for the LTP_PREGS induction and was sensitive to Ifenprodil. These results suggest that PREGS induces an acute increase in the NR2B tyrosine phosphorylation which enhances the Ca²⁺ influx through NMDAr, followed by an activation of the ERK/CREB signaling cascade that leads to LTP_PREGS.