In the brain and heart, auxiliary KChIPs coassemble with pore-forming Kv4 α- subunits to form a native channel complex and regulate the expression and gating properties of Kv4 currents as well as membrane excitability. Among the KChIP1-4, KChIP4a exhibits a unique N-terminus that is known to suppress Kv4 function, but the underlying mechanism for Kv4 inhibition remains unknown. In this study, using approaches of confocal imaging, biochemistry and electrophysiology, we identified the hydrophobic and aliphatic residues 12-17, LIVIVL within the KChIP4a N-terminus, that function as a novel ER retention motif to reduce surface expression of Kv4-KChIP channel complex. The ability of LIVIVL motif to retain Kv4 proteins is transferable and depends on its flanking location, but not buried in the middle of protein sequence. Interestingly, adjacent to the ER retention motif, the residues 19-21 (VKL motif) directly interact with Kv4.3 to enhance closed-state inactivation (CSI) and lead to Kv4.3 current inhibition. Because that auxiliary KChIPs can alter biophysical and physiological properties of Kv4 channels by regulating subunit composition of native Kv4 channel complex, we then analyzed the expression profile of two KChIP4 splice variants KChIP4a and KChIP4bl that have opposite effects on Kv4 function in rat dorsal root ganglion (DRG) neurons under pain conditions. Our findings show that KChIP4a mRNA was down-regulated in L4-5 DRG neurons 7 days after spinal nerve ligation in rats that received chloral hydrate (0.3 g/kg, intraperitoneally) before operation. Taken together, our findings reveal two distinct mechanisms by which KChIP4a suppresses Kv4 function through ER retention and promoting CSI. Differential regulation of auxiliary KChIPs expression in DRG neurons are likely involved in development of pathological pain conditions.