Porcine small intestinal epithelial cell line (IPEC-J2) is a good research model exploring the impact of feed additives on intestinal epithelial cells. Monobutyrin (MB), as a derivative of butyric acid (BA), overcomes the shortcomings of BA. MB can maintain intestinal barrier function in animals, but its underlying regulatory mechanism is unknown. Thus, we used IPEC-J2 cells as the research object. We were using real-time fluorescence quantitative PCR, western blot, immunofluorescence, and transcriptomics technology to explore the effect of MB on the barrier function of IPEC-J2 cells and its regulatory mechanism. The results found that MB treatment could cause IPEC-J2 cells to occur a response to hypoxia at the transcriptional level, thereby increasing the expression of hypoxia-inducible factor 1 and phospho-extracellular signal-regulated kinase 1/2 protein and improving the expression of tight junction proteins. Therefore, MB can alleviate the activation of the NF-κB signaling pathway. In addition, MB mitigates the damage to cell transmembrane glycoproteins, microvilli, and tight junctions caused by lipopolysaccharides (LPS) stimulation, thus resisting the effects of LPS. As a dietary supplement, MB has good application prospects in maintaining the intestinal epithelial barrier function of animals.