Kv4 (Shal) potassium channels are responsible for the transient outward K(+) currents in mammalian hearts and central nervous systems. Heteropoda toxin 2 (HpTx2) is an inhibitor cysteine knot peptide toxin specific for Kv4 channels that inhibits gating of Kv4.3 in the voltage-dependent manner typical for this type of toxin. HpTx2 interacts with four independent binding sites containing two conserved hydrophobic amino acids in the S3b transmembrane segments of Kv4.3 and the closely related Kv4.1. Despite these similarities, HpTx2 interaction with Kv4.1 is considerably less voltage-dependent, has smaller shifts in the voltage-dependences of conductance and steady-state inactivation, and a 3-fold higher K(d) value. Swapping four nonconserved amino acids in S3b between the two channels exchanges the phenotypic response to HpTx2. To understand these differences in gating modification, we constructed Markov models of Kv4.3 and Kv4.1 activation gating in the presence of HpTx2. Both models feature a series of voltage-dependent steps leading to a final voltage-independent transition to the open state and closely replicate the experimental data. Interaction with HpTx2 increases the energy barrier for channel opening by slowing activation and accelerating deactivation. The greater degree of voltage-dependence in Kv4.3 occurs because it is the voltage-dependent transitions that are most affected by HpTx2; in contrast, it is the voltage-independent step in Kv4.1 that is most affected by the presence of toxin. These data demonstrate the basis for subtype-specificity of HpTx2 and point the way to a general model of gating modifier toxin interaction with voltage-gated ion channels.