Melittin (MLT), as a natural active biomolecule, can penetrate the tumor cell membrane to play a role in cancer treatment and will attract more attention in future development of antitumor drugs. The main component of natural bee venom MLT was modified by introducing a pH-sensitive amide bond between the 2,3-dimethyl maleimide (DMMA) and the lysine (Lys) of MLT (MLT-DMMA). MLT and its corresponding modified peptide MLT-DMMA were used for antitumor and biocompatibility validation. The biomaterial characteristics were tested by MALDI-TOF MS, 1H NMR, IUPAC and HPLC, cell viability, hemolytic and animal experiment safety evaluation. Compared with the primary melittin, the modified peptide showed decreased surface charge and low cytotoxicity in physiological conditions. Moreover, cell assays confirmed the acid-activated conversion of amide bond resulting in adequate safety during delivery and timely antitumor activity in tumor lesions. Thus, MLT-DMMA provided a feasible platform to improve the targeted and safe antitumor applications.