Kv4/KChIP potassium channels are a major class of rapidly inactivating K⁺ channels in neurons and cardiac muscle. Modulation of Kv4/KChIP channels by polyunsaturated fatty acids (PUFAs) is important in the regulation of cellular excitability and the induction of activity-dependent synaptic plasticity. Using the Xenopus oocyte expression system, we studied the inhibition by PUFAs of the peak outward K⁺ current and the accompanying increase in the rate of current inactivation of rKv4.2/rKChIP1b. Inhibitory effects do not depend on KChIP co-expression since Kv4.2 channels lacking an N-terminal KChIP association region were substantially inhibited by PUFAs and showed strong kinetic modulation. PUFAs accelerated both the fast and slow time constants that describe the kinetics of Kv4/KChIP inactivation. The time course of entry into closed inactivated states was facilitated by PUFAs but steady-state inactivation and recovery from inactivation were unaltered. PUFA inhibition of Kv4/KChIP current was not use-dependent. The concentration-response relationship for AA inhibition of Kv4/KChIP channels mimicked that for activation of TRAAK channels. Internal serum albumin largely prevents the inhibitory effects of externally applied AA and the membrane impermeant AA-CoA is inactive when applied externally. Overall, our data suggest that PUFAs inhibit Kv4/KChIP channels by facilitating inactivation of open and closed channels and access of the fatty acid to the internal leaflet of the membrane is important. These results improve our understanding of the mechanisms for the inhibitory effects of PUFAs on Kv4/KChIP channel function.