Many scorpion toxins that act on sodium channels (NaScTxs) have been characterized till date. These toxins may act modulating the inactivation or the activation of sodium channels and are named α- or β-types, respectively. Some venom toxins from Tityus obscurus (Buthidae), a scorpion widely distributed in the Brazilian Amazon, have been partially characterized in previous studies; however, little information about their electrophysiological role on sodium ion channels has been published. In the present study, we describe the purification, identification and electrophysiological characterization of a NaScTx, which was first described as Tc54 and further fully sequenced and renamed To4. This toxin shows a marked β-type effect on different sodium channel subtypes (hNav1.1–hNav1.7) at low concentrations, and has more pronounced activity on hNav1.1, hNav1.2 and hNav1.4. By comparing To4 primary structure with other Tityus β-toxins which have already been electrophysiologically tested, it is possible to establish some key amino acid residues for the sodium channel activity. Thus, To4 is the first toxin from T. obscurus fully electrophysiologically characterized on different human sodium channel isoforms.