Aflatoxin B1 (AFB₁) and aflatoxin M₁ (AFM₁) are natural mycotoxins that frequently found in food and feed. Numerous reports have indicated that these toxins pose serious risks to human and animal health. However, there are few data in the literature regarding the impairment of AFB₁ and AFM₁ on intestine. The present study therefore was undertaken to investigate the cytotoxic effect and DNA damage of these toxins on human colon carcinoma cell line Caco-2, especially the differentiated cells that resemble mature small intestinal enterocytes. Both undifferentiated (UC) and differentiated (DC) cell were treated with AFB₁ and AFM₁ at various concentrations for 24, 48 and 72 h. Cell viability, lactate dehydrogenase (LDH) release and reactive oxygen species (ROS) production were determined, and DNA damage was accessed by comet assay. Statistical analysis of data was carried out using SAS9.2, statistical software package. Data showed that AFB₁ and AFM₁ inhibited UC and DC cell growth and increased the LDH release in a time- and dose-dependent manner. Besides, AFB₁ treatment resulted in an evident increase in cytotoxicity over AFM₁ at the high dosage (P < 0.05). Moreover, DC were more sensitive to toxins compared with UC (P < 0.05), particularly after exposure of 72 h at the dose of 1 μg/mL, as indicated from the lower cell viability(48% vs. 67%, AFB₁ treatment) and the higher LDH release (118% vs. 186%, AFM₁ treatment; 142% vs. 194%, AFB₁ treatment). Marked impacts in the genetic damage were observed after treatment with 2 toxins on UC and DC, even higher than those of H₂O₂ treatment (positive control). Compared with UC, DC also had more DNA damage (P < 0.05), which might due to the alteration of cells during differentiation. All these cytotoxic effects might associate with the strong intracellular ROS generation in the presence of toxins. The present study provided the first experimental evidence of the in vitro DNA damage of DC induced by AFB1 and AFM1.