α-synuclein, which is a major component of Lewy bodies, has been linked to the pathogenesis of Parkinson's disease and dementia with Lewy bodies. Despite increasing evidence suggests that soluble oligomers rather than insoluble mature fibrils, are the pathogenic species, how the soluble α-synuclein oligomers accumulate intracellularly and alter neuronal activities into pathological condition, has not been studied directly. We examined the action of α-synuclein on neuronal excitabilities by injecting α-synuclein protein into pyramidal neurons in mouse frontal cortical slices through whole-cell patch pipettes. Western blot analysis showed that the pipette solution including α-synuclein incubated with dopamine for 3 days (α-SN+DA), had higher order oligomer in comparison with α-synuclein-containing solution without dopamine (α-SN). Intracellular application of α-SN+DA significantly suppressed spike firing during current injection compared with α-SN, but did not altered resting membrane potential, spike width, spike after depolarization, and H current. Nimodipine and apamin blocked the effect of α-SN+DA on spike firing. α-SN+DA prolonged the duration of spike afterhyperpolarization (AHP) and increased the charge transfer carried by the current elicited by depolarization pulses that would produce AHP under current clamp (I_AHP), both of which were inhibited by apamin. These results suggest that α-synuclein oligomers reduce spike frequency by enhancing AHP, which may cause the downregulation of neocortical activities in Parkinson's disease and dementia with Lewy bodies.