Scorpion toxins can kill other animals by inducing paralysis and arrhythmia, which limits the potential applications of these agents in the clinical management of diseases. Antitumor-analgesic peptide (AGAP), purified from Karsch, has been proved to possess analgesic and antitumor activities. Trp, a conserved aromatic residue of AGAP, might play an important role in mediating AGAP activities according to the sequence and homology-modeling analyses. Therefore, an AGAP mutant, W38G, was generated, and effects of both AGAP and the mutant W38G were examined by whole-cell patch clamp techniques on the sodium channels hNa1.4 and hNa1.5, which were closely associated with the biotoxicity of skeletal and cardiac muscles, respectively. The data showed that both W38G and AGAP inhibited the peak currents of hNa1.4 and hNa1.5; however, W38G induced a much weaker inhibition of both channels than AGAP. Accordingly, W38G exhibited much less toxic effect on both skeletal and cardiac muscles than AGAP The analgesic activity of W38G and AGAP were verified as well, and W38G retained analgesic activity similar to AGAP. Inhibition to both Na1.7 and Na1.8 was involved in the analgesic mechanism of AGAP and W38G. These findings indicated that Trp was a key amino acid involved in the biotoxicity of AGAP, and the AGAP mutant W38G might be a safer alternative for clinical application because it retains the analgesic efficacy with less toxicity to skeletal and cardiac muscles.