Di-butyl phthalate (DBP) is a major type of phthalate (PAE) contaminant widely used as a plasticizer. Its environmental presence poses threat to humans and aquatic organisms. In this study, Ctenopharyngodon idellus kidney (CIK) cell model was exposed to 100 μM DBP to investigate its effects. Apoptosis was assessed using acridine orange/ethidium bromide staining, flow cytometry, and fluorescein isothiocyanate/propidium iodide labeling. DBP exposure increased the percentage of apoptotic cells. Activities of antioxidant enzymes, superoxide dismutase, total antioxidant activity, glutathione peroxidase, and catalase were inhibited by DBP, whereas the levels of peroxide products were increased. Heat shock proteins were upregulated as a defense mechanism against DBP-induced stress. Further analysis revealed that the Pi3k/Akt pathway, which regulates physical processes to protect cell function, was suppressed by DBP exposure. Reverse transcription-quantitative PCR and western blotting revealed that DBP inhibited Pi3k/Akt signaling while apoptosis gene expression was increased. Notably, these effects of heat shock proteins and Pi3k/Akt, were reversed by N-acetylcysteine. In conclusion, DBP accelerates apoptosis of CIK cells by inhibiting the Pi3k/Akt pathway and anti-oxidative enzyme activities, promoting reactive oxygen species accumulation and enhancing peroxide product generation. These findings highlight the cytotoxic effects of DBP and underscore the need for further research. Our results provide a foundation for further study.