Neuregulin-2 (NRG2) has been identified as a member of the family of growth factor that contains an EGF-like domain with its domain structure similar to NRG1. NRG1 and NRG2 are expressed in distinct cell populations in the brain. Unlike the expression of NRG-1 which is sporadic but widespread over the brain, NRG2 is expressed in a few restricted regions in the brain including the hippocampal dentate granule cell (GC) layer. NRG2 has been implicated in proliferation of neural stem cells in the subventricular zone, but it is little understood whether NRG2 plays a role in synaptogenesis, which is essential for newborn neurons to be integrated into a preexisting neural circuit. Using retrovirus encoding GFP and RNAi against NRG2, we studied the role of NRG2 in development of GFP-labeled newborn GCs in rat organotypic hippocampal culture. To study the role of NRG2 in synpatogenesis, we infected the organotypic cultured hippocampal slice with retrovirus encoding NRG2-targeting miRNA cloned downstream of tetracycline-response element (TetOn-miNRG-2). Newborn GCs infected with such retrovirus displayed significantly lower expression of NRG2 after treatment with doxycycline (dox) than non-treated newborn GCs as a control. To assess the synaptic development in newborn GCs, we recorded GABAergic or glutamatergic postsynaptic current (GPSC or EPSC) induced by stimulation of the perforant pathway. Under the control conditions, the amplitude of GPSC started to increase from 7th day post-infection (7dpi) and reached a plateau level at 10dpi. Treatment of doxycycline from 4dpi abolished such a steep increase in the GPSC amplitude. Accordingly, immunohistochemistry using anti-VGAT revealed that the number of GABAergic presynaptic terminals on the soma of newborn GCs was lower in the dox-treated hippocampal slice than the control slice. In contrast, dox-treatment from 7dpi or 10dpi displayed no significant effect on GPSC, suggesting NRG2 plays essential role in GABAergic synapse formation at 4~7dpi but not in its maintenance after 7dpi. The amplitude of AMPA and NMDA receptor-mediated excitatory postsynaptic currents (AMPA-EPSC and NMDA-EPSC, respectively) started to steeply increase from 13dpi. We studied the role of NRG2 in glutamatergic synapse formation by treating the culture with dox from 7dpi, which had a limited effect on GPSC. The amplitudes of AMPA- and NMDA-EPSCs were significantly lower in dox-treated newborn GCs compared with control GCs. Furthermore, the ratio of AMPA- to NMDA-EPSC was reduced by dox-treatment from 7dpi, implying that NRG2 contributes to the maturation of excitatory synapses. These results indicate that the NRG2 expression in newborn GCs is essential for both GABAergic and glutamatergic synaptogenesis.