To study the effect of chitooligosaccharides(COS) on aflatoxin B1(AFB₁)-induced toxic injury of rat liver cells (BRL 3A cells). The CCK-8 method was used to determine the IC₅₀ value of AFB₁ on cells and the non-damage concentration of COS on cells. After COS pretreated the cells for 6 hours and then adding AFB₁ to continue culturing for 24 hours, we used the kit to determine the cell viability, reactive oxygen species (ROS) levels, malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, glutathione S-transferase (GST) activity and apoptosis rate. The mRNA relative expression levels of Nrf2, Keap1, Ho-1, Nqo1, Bax and Bcl-2 genes were determined by real-time quantitative PCR (RT qPCR), and the hierarchical clustering and enrichment pathways of differentially expressed genes were analyzed by RNA-seq. Results: The IC₅₀ of AFB₁ to BRL 3A cells was 15.86 μmol/L, and the COS concentration less than 125 μmol/L would not cause toxic damage to the BRL 3A cells. COS can reduce the increase of ROS level and MDA content in cells induced by AFB₁, enhance the activity of SOD and GST, thereby improve the cell's own antioxidant capacity and reduce the rate of apoptosis. AFB₁ can cause the significant expression of the pro-apoptotic gene Bax (P<0.05), and significantly reduce the transcription levels of Nrf2, Keap1, Ho-1, and Nqo1 (P<0.05), while COS pretreatment can significantly increase the expression of Nrf2, Keap1, Ho-1, Nqo1(P<0.05), and significantly reduce the expression level of Bax (P<0.05). In the results of RNA-seq enrichment pathways, COS may also alleviate the cytotoxic damage induced by AFB₁ through the metabolism of xenobiotics by cytochrome P450, drug metabolism-cytochrome P450 and p53 signaling pathway (P<0.05). In summary,COS pretreatment can intervene the toxic injury of BRL 3A cells induced by AFB₁, which may be mediated by Nrf2 signaling pathway, metabolism of xenobiotics by cytochrome P450, drug metabolism-cytochrome P450 and p53 signaling pathway.