Grape seed extract (GSE), a rich source of polyphenols, is reported to possess antioxidant, anti-inflammatory and immunomodulatory properties. The objective of the present study was to determine whether GSE could attenuate the heat stress-induced responses of jejunum epithelial cells (JEC) in cattle. The JEC of a steer (Simmental × Qinchuan) were exposed to heat stress for 2 h in the absence (0 μg/ml) or presence (10, 20, 40 and 80 μg/ml) of GSE in the culture medium. When cultured at 40°C, JEC supplemented with GSE exhibited increased glutathione peroxidase activity (P= 0·04), viability (P= 0·004), and mRNA expression of epidermal growth factor (EGF; P= 0·03) and EGF receptor (EGFR; P= 0·01). Under the same conditions, the cells exhibited decreased mRNA expression of IL-8 (P= 0·01) and TNF-α (P= 0·03) and decreased protein concentrations of IL-1β (P= 0·02), Toll-like receptor 4 (TLR4; P= 0·04) and heat shock protein 70 (HSP70; P< 0·001). When cultured at 43°C, JEC supplemented with GSE exhibited increased catalase activity (P= 0·04), viability (P< 0·001), and mRNA expression of EGF (P< 0·001) and EGFR (P< 0·001) and decreased protein concentrations of IL-1β (P< 0·001), TLR4 (P= 0·03) and HSP70 (P< 0·001), as well as mRNA expression of IL-8 (P< 0·001), TLR4 (P= 0·002) and TNF-α (P< 0·001). Temperature × GSE concentration interactions were also observed for the concentrations of IL-1β (P< 0·001), IL-8 (P< 0·001), TNF-α (P= 0·01) and HSP70 (P= 0·04) and viability (P< 0·001) of JEC. The results of the present study indicate that GSE can attenuate the responses of JEC induced by heat stress within a certain range of temperatures.