The present study aimed to investigate the effect of glutamine (Gln) addition on the damage of porcine intestinal epithelial cells (IPEC-J2) induced by heat stress (HS). IPEC-J2 cultured in logarithmic growth period in vitro were firstly exposed to 42 °C for 0.5, 1, 2, 4, 6, 8, 10, 12, and 24 h for cell viability and cultured with 1, 2, 4, 6, 8, or 10 mmol Gln per L of culture media for heat shock protein 70 (HSP70) expression to determine the optimal disposal strategy (HS, 42 °C for 12 h and HSP70 expression, 6 mmol/L Gln treatment for 24 h). Then IPEC-J2 cells were divided into three groups: control group (Con, cultured at 37 °C), HS group (HS, cultured at 42 °C for 12 h), and glutamine group (Gln+HS, cultured at 42 °C for 12 h combined with 6 mmol/L Gln treatment for 24 h). The results showed that HS treatment for 12 h significantly decreased the cell viability of IPEC-J2 (P < 0.05) and 6 mmol/L Gln treatment for 12 h increased HSP70 expression (P < 0.05). HS treatment increased the permeability of IPEC-J2, evidenced by the increased fluorescent yellow flux rates (P < 0.05) and the decreased transepithelial electrical resistance (P < 0.05). Moreover, the downregulated protein expression of occludin, claudin-1, and zonula occludens-1 was observed in HS group (P < 0.05), but Gln addition alleviated the negative effects on permeability and the integrity of intestinal mucosal barrier induced by HS (P < 0.05). In addition, HS resulted in the elevations in HSP70 expression, cell apoptosis, cytoplasmic cytochrome c potential expression, and the protein expressions of apoptosis-related factors (apoptotic protease-activating factor-1, cysteinyl aspartate-specific proteinase-3, and cysteinyl aspartate-specific proteinase-9) (P < 0.05); however, the reductions in mitochondrial membrane potential expression and B-cell lymphoma-2 expression were induced by HS (P < 0.05). But Gln treatment attenuated HS-induced adverse effects mentioned above (P < 0.05). Taken together, Gln treatment exhibited protective effects in protecting IPEC-J2 from cell apoptosis and the damaged integrity of epithelial mucosal barrier induced by HS, which may be associated with the mitochondrial apoptosis pathway mediated by HSP70.