Cardiotoxin III (1), a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, has potential therapeutic activity in cancer. Treatment with 1 reduced phosphorylation of EGFR and Akt, as well as ERK in Ca9-22 cells. Moreover, 1-treatment inhibited constitutive activation of STAT3 and STAT5 in a time-dependent manner. Up-regulation of Bax and down-regulation of anti-apoptotic proteins including Bcl-2, Bcl-X(L), and myeloid cell leukemia-1(Mcl-1) were also found in cells treated with 1. In addition, 1-treatment disrupted mitochondrial membrane potential (DeltaPsim) and resulted in release of mitochondrial cytochrome c and activation of both caspases-9 and -3. AG1478, a specific pharmacological inhibitor of EGFR activation, mimics the cytotoxic effects of 1. Taken together, these results showed that 1 causes significant induction of apoptosis in Ca9-22 cells via abolition of the EGFR-mediated survival pathway of these cells. Thus, cardiotoxin III appears to be a potential therapeutic agent for killing oral squamous carcinoma Ca9-22 cells.