The nucleus accumbens (NAc) receives excitatory projections from various regions in the cerebral cortex. The insular and prefrontal cortices are principal sorce of projections to the NAc. Here, we examined the projection patterns and their properties by whole-cell patch-clamp recordings from medium spiny neurons (MSNs) and cholinergic neurons (ChNs) in the NAc in combination with an optogenetic technique. Photostimulation of IC fibers elicited EPSCs only in medium spiny neurons (MSNs), but not in ChNs. On the other hand, photostimulation of mPFC fibers elicited EPSCs both in MSNs and ChNs. Bath application of carbachol (10 μM) effectively suppressed the amplitude of photostimulation-evoked EPSCs recorded from MSNs. Pirenzepine (10 μM) counteracted this cholinergic effect. These results suggest that the IC and mPFC differentially regulate NAc activities by targeting different neural subtypes, MSNs and ChNs: projections from the mPFC but not from the IC are suppressed by M1 receptor activation in the NAc.