In this study, whole-cell recording was carried out to explore the effects of 17β-estradiol on voltage-gated K+ (Kv) currents in N2A cells. The acute exposure to 17β-estradiol, in a concentration-dependent manner, significantly inhibited the peak and steady-state currents through Kv channels, showing IC50 values of 3.6 and 3.8 μM, respectively. The reduction in both the amplitude and the decay rate of Kv currents, with an increase in depolarization, suggested that it was a voltage-dependent block. The activation and inactivation experiments were conducted to determine the exact causes of the inhibitory effects. The half-maximum activation potential (V1/2) was +8.1 mV in control and remained stable after exposure to 10 μM 17β-estradiol. For steady-state inactivation, the half-maximum inactivation potential (V1/2) was −45.0 mV and shifted right to −39.7 mV without a statistical difference, and the time constants of recovery from inactivation were not altered by 17β-estradiol, suggesting that the depression was not correlated with the inactivation gate.