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Inhibition of T-type calcium channels (Ca_V3) prevents development of diseases related to cardiovascular and nerve systems. Further, knockout animal studies have revealed that some diseases are mediated by specific subtypes of Ca_V3. However, subtype-specific Ca_V3 inhibitors for therapeutic purposes or for studying the physiological roles of Ca_V3 subtypes are missing. To bridge this gap, we employed our spider venom library and uncovered that <i>Avicularia spec.</i> (“Amazonas Purple”, Peru) tarantula venom inhibited specific T-type Ca_V channel subtypes. By using chromatographic and mass-spectrometric techniques, we isolated and sequenced the active toxin ω-Avsp1a, a C-terminally amidated 36 residue peptide with a molecular weight of 4224.91 Da, which comprised the major peak in the venom. Both native (4.1 μM) and synthetic ω-Avsp1a (10 μM) inhibited 90% of Ca_V3.1 and Ca_V3.3, but only 25% of Ca_V3.2 currents. In order to investigate the toxin binding site, we generated a range of chimeric channels from the less sensitive Ca_V3.2 and more sensitive Ca_V3.3. Our results suggest that domain-1 of Ca_V3.3 is important for the inhibitory effect of ω-Avsp1a on T-type calcium channels. Further studies revealed that a leucine of T-type calcium channels is crucial for the inhibitory effect of ω-Avsp1a.