The mammalian Kv4.3 potassium channel is a fast activating and inactivating K+ channel widely distributed in mammalian tissues. Kv4.3 is the major component of various physiologically important currents ranging from A-type currents in the CNS to the transient outward potassium conductance in the heart (I(to)). Here we show that the KCNE3 beta-subunit has a strong inhibitory effect on current conducted by heterologously expressed Kv4.3 channels. KCNE3 reduces the Kv4.3 current amplitude, and it slows down the channel activation and inactivation as well as the recovery from inactivation. KCNE3 also inhibits currents generated by Kv4.3 in complex with the accessory subunit KChIP2. We find the inhibitory effect of KCNE3 to be specific for Kv4.3 within the Kv4 channel family. Kv4.3 has previously been shown to interact with a number of beta-subunits, but none of the described subunit-interactions exert an inhibitory effect on the Kv4.3 current.