2,6‐dichloro‐1,4‐benzoquinone (2,6‐DCBQ), as an emerging disinfection by‐product, has been frequently detected in waters, posing potential health risk on public health. Although some studies have pointed out that 2,6‐DCBQ exposure can induce cytotoxicity, limited information is available for underlying mechanism for 2,6‐DCBQ‐induced cytotoxicity. To explore this mechanism, we assessed the levels of reactive oxygen species (ROS), acridine orange (AO) staining, and the mRNA transcriptions of genes (Chk2, Cdk2, Ccna, Ccnb and Ccne) involved in cell‐cycle and genes (p53, bax, bcl‐2 and caspase 3) involved in apoptosis in zebrafish embryo, after exposed to different concentrations (10, 30, 60, 90 and 120 μg/L) of 2,6‐DCBQ for 72 h. Our results indicated that 2,6‐DCBQ exposure induced ROS generation and cell apoptosis, and disturbed the mRNA transcription of genes related to cell cycle and apoptosis in zebrafish embryo. Moreover, we also found that 30 ~ 60 μg/L 2,6‐DCBQ is the important transition from cell‐cycle arrest to cell apoptosis. These results provided novel insight into 2,6‐DCBQ‐induced cytotoxicity.