Crotamine, a 5-kDa peptide, possesses a unique biological versatility. Not only has its cell-penetrating activity become of clinical interest but, moreover, its potential selective antitumor activity is of great pharmacological importance. In the past, several studies have attempted to elucidate the exact molecular target responsible for the crotamine-induced skeletal muscle spasm. The aim of this study was to investigate whether crotamine affects voltage-gated potassium (K_V) channels in an effort to explain its in vivo effects. Crotamine was studied on ion channel function using the two-electrode voltage clamp technique on 16 cloned ion channels (12 K_V channels and 4 Na_V channels), expressed in Xenopus laevis oocytes. Crotamine selectively inhibits K_V1.1, K_V1.2, and K_V1.3 channels with an IC₅₀ of ∼300 nM, and the key amino acids responsible for this molecular interaction are suggested. Our results demonstrate for the first time that the symptoms, which are observed in the typical crotamine syndrome, may result from the inhibition of K_V channels. The ability of crotamine to inhibit the potassium current through K_V channels unravels it as the first snake peptide with the unique multifunctionality of cell-penetrating and antitumoral activity combined with K_V channel-inhibiting properties. This new property of crotamine might explain some experimental observations and opens new perspectives on pharmacological uses.