Rapidly activating and inactivating A-type K+ currents (Ia) encoded by Kv4.2 and Kv4.3 pore-forming (a) subunits of the Kv4 subfamily are key regulators of neuronal excitability. Previous studies have suggested a role for Kv4.1 a-subunits in regulating the firing properties of mouse suprachiasmatic nucleus (SCN) neurons. To test this, we utilized an RNA-interference strategy to knockdown Kv4.1, acutely and selectively, in the SCN. Current-clamp recordings revealed that the in vivo knockdown of Kv4.1 significantly (p < 0.0001) increased mean ± SEM repetitive firing rates in SCN neurons during the day (6.4 ± 0.5 Hz) and at night (4.3 ± 0.6 Hz), compared with nontargeted shRNA- expressing SCN neurons (day: 3.1 ± 0.5 Hz; night: 1.6 ± 0.3 Hz). Ia was also significantly (p < 0.05) reduced in Kv4.1 -targeted shRNA-expressing SCN neurons (day: 80.3 ±11.8 pA/pF; night: 55.3 ± 7.7 pA/pF). compared with nontargeted shRNA-expressing (day: 121.7 ± 10.2 pA/pF; night: 120.6 ± 16.5 pA/pF) SCN neurons. The magnitude of the effect of Kv4.1 -targeted shRNA expression on firing rates and lA was larger at night. In addition, Kv4.1 –targeted shRNA expression significantly ip < 0.001) increased mean ± SEM nighttime input resistance (Rin; 2256 ±166 Mil), compared to nontargeted shRNA-expressing SCN neurons (1143 ± 93 MΩ). Additional experiments revealed that acute knockdown of Kv4.1 significantly (p < 0.01) shortened, by ~0.5 h, the circadian period of spontaneous electrical activity, clock gene expression and locomotor activity demonstrating a physiological role for Kv4.1-encoded lA channels in regulating circadian rhythms in neuronal excitability and behavior. © 2017 Hermanstyne et al.