Antimicrobial peptides have received increasing attention as potential antitumor drugs due to their new mode of action. However, the systemic toxicity at high concentration always hampers their successful utilization for tumor therapy. Here, we designed a new type of acid-activated antimicrobial peptide AMitP by conjugating antimicrobial peptide MitP to its anionic binding partner MitPE via a disulfide linker. Compared with MitP, AMitP displayed significant antitumor activity at acidic pH and low cytotoxicity at normal pH. The results of MD simulations demonstrate that the changes of structure and membrane binding tendency of AMitP at different pH values played an important role in its pH-dependent antitumor activity. In addition, AMitP showed significant enzymatic stability compared with MitP, suggesting a potential for in vivo application. In short, our work opens a new avenue to develop antimicrobial peptides as potential antitumor drugs with high selectivity.