Tarantula venom glands produce a large variety of bioactive peptides. Chilean common tarantula Grammostola rosea has relatively large venom glands, and the secreted venom and the venom glands have been utilized in both proteomic and genomic approaches, respectively. The expressed sequence tag analysis for the cDNA library has unveiled 48 toxin-like peptides from the tarantula to date. Among them, 24 toxins are inhibitor cystine knot motif peptides (GTx1 and GTx2), 11 peptides (GTx3) are mamba intestinal toxin 1 (MIT1)-like peptides, and 7 are GTx4s similar to ESTX-like peptides identified from another tarantula species. Peptides similar to JZTX-64, aptotoxin, CRISP, or TCTP were also identified. GTx3 series possess a cysteine framework that is conserved among vertebrate MIT1, Bv8, prokineticins, and invertebrate astakines, while these peptides are reported to have diverse bioactivities such as muscle contractions, angiogenesis, etc. GTx-CRISP is the first CRISP-like protein identified from the arthropod vemon. A novel peptide, named GTx1-15, shows 76.5 % sequence homology with phrixotoxin 3(PaurTx3) isolated from another spider. PaurTx3 was characterized to block sodium channels; however, GTx1-15 preferentially inhibited T-type voltage-dependent calcium channels. Secondary and tertiary structure prediction in silico revealed that GTx1-15 and PaurTx3, and other sodium channel blockers such as hainantoxin-IV and ceratotoxin 2 as well, show very similar β-strand composition, distribution of optimal docking areas, and surface electrostatic potential. The findings may suggest that these peptide toxins evolved from common ancestors by gene duplication under evolution pressures to maintain surface environment appropriate for targeting low-voltage-dependent ion channels.