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Tilapia piscidin 4 (TP4) is a 25-amino-acid cationic antimicrobial peptide derived from Nile tilapia (Oreochromis niloticus). TP4 has demonstrated strong antimicrobial activity against a broad range of pathogens and exhibits additional biological functions, including wound healing and anticancer activity. In a previous study, recombinant TP4 peptide was produced in the Pichia pastoris KM71H expression system and purified by Ni-NTA affinity chromatography. The aim of this study was to evaluate and compare the cytotoxicity of recombinant TP4 against HT-29 colon cancer and HGF-1 healthy gingival fibroblast cells.The antimicrobial activity of TP4 was first assessed using minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays, confirming its efficacy. Subsequently, the cytotoxic effects of TP4 on HT-29 and HGF-1 cells were further analyzed using the MTT, lactate dehydrogenase (LDH), total antioxidant/oxidant status (TAS/TOS), and malondialdehyde (MDA) assays. TP4 disrupted the antioxidant balance in both cell types, with a significant increase in the oxidant levels and a marked decrease in the antioxidant levels in HT-29 cells. The IC₅₀ values of TP4 were determined to be 15 μg/mL for HT-29 cells and 25 μg/mL for HGF-1 cells. Apoptosis assays, including Annexin V & Dead Cell, RT-qPCR, and ELISA, revealed that TP4 induced apoptosis predominantly in HT-29 cells, as evidenced by the upregulation of BAX and CASP3 and the downregulation of the BCL-2. In conclusion, TP4 exhibited selective and potent apoptotic effects on HT-29 colon cancer cells, suggesting its potential as a novel therapeutic agent for colon cancer treatment.