Spider venoms, despite their toxicity, represent rich sources of pharmacologically active compounds with biotechnological potential. However, in view of the large diversity of the spider species, the full potential of their venom molecules is still far from being known. In this work, we report the purification and structural and functional characterization of GiTx1 (β/κ-TRTX-Gi1a), the first toxin purified from the venom of the Brazilian tarantula spider Grammostola iheringi. GiTx1 was purified by chromatography, completely sequenced through automated Edman degradation and tandem mass spectrometry and its structure was predicted by molecular modeling. GiTx1 has a MW of 3.585 Da, with the following amino acid sequence: SCQKWMWTCDQKRPCCEDMVCKLWCKIIK. Pharmacological activity of GiTx1 was characterized by electrophysiology using whole-cell patch clamp on dorsal root ganglia neurons (DRG) and two-electrode voltage-clamp on voltage-gated sodium and potassium channels subtypes expressed in Xenopus laevis oocytes. GiTx1, at 2 μM, caused a partial block of inward (∼40%) and outward (∼20%) currents in DRG cells, blocked rNav1.2, rNav1.4 and mNav1.6 and had a significant effect on VdNav, an arachnid sodium channel isoform. IC50 values of 156.39 ± 14.90 nM for Nav1.6 and 124.05 ± 12.99 nM for VdNav, were obtained. In addition, this toxin was active on rKv4.3 and hERG potassium channels, but not Shaker IR or rKv2.1 potassium channels. In summary, GiTx1 is a promiscuous toxin with multiple effects on different types of ion channels.