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 Buthus martensii Karsch (BmK), has been proved to possess analgesic and antitumor activities. Trp38, 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 examine by whole-cell patch clamp techniques on the sodium channels hNav1.4 and hNav1.5, which were closely associated to the biotoxicity of skeletal and cardiac muscles, respectively. The data showed both W38G and AGAP inhibited the peak currents of hNav1.4 and hNav1.5, however, W38G induced a much weaker inhibition to both channels than AGAP. Accordingly, W38G exhibited much less toxic to both skeletal and cardiac muscles than AGAP in vivo. The analgesic activity of W38G and AGAP were verified in vivo as well and W38G retained analgesic activity similar to AGAP. Inhibition to both Nav1.7 and Nav1.8 was involved in the analgesic mechanism of AGAP and W38G. These findings indicated that Trp38 was a key amino acid involved in the biotoxicity of AGAP, and the AGAP mutant W38G might be a safer alternative for clinical application since it retained the analgesic efficacy with less toxicity to skeletal and cardiac muscles.