The purpose of this study was to investigate structure of Hemp seed polysaccharide (HSP) and the protective effect of HSP from HO-induced oxidative damage in IPEC-1 cells and the possible mechanism of this protection. Analysis of monosaccharide composition and structure of two fractions HSPand HSPfrom polysaccharide of Hemp seed (HSPc) were analyzed by high performance liquid chromatography (HPLC) and Fourier transform infrared spectroscopy (FT-IR). The results showed that both HSPand HSPcontain sulfate groups, which are sulfated polysaccharides. In IPEC-1 cells model, the release of LDH and MDA was significantly decreased, and the activities of SOD, GSH-Px and CAT were significantly increased in HSPand HSP-treated group. HSPdramatically increased the gene expression of antioxidant enzymes and phase II detoxification enzymes measured by real-time fluorescent quantitative reverse transcription-polymerase chain reaction (qRT-PCR). In addition, HSPup-regulated the expression level of intracellular transcription factor Nuclear factor erythroid-2-related factor 2 (Nrf2) and inhibited the level of Kelch-like ECH-associated protein 1 (Keap1) with Western blot analysis. Collectively, the present study suggested that HSPhas the protective effect of IPEC-1 cells against HO-induecd oxidative stress. This protection mechanism may be related to activation of the Keap1/Nrf2 signaling pathway.