The objective of this study was to determine the role of SLC15A4 in the muramyl dipeptide (MDP)-mediated inflammatory response of bovine rumen epithelial cells (BRECs).  First, changes in the mRNA expression of pro-inflammatory factor genes in BRECs following 10 μg mL^–1 MDP treatments were examined.  RT-qPCR results showed that the expression of pro-inflammatory factor (IL-1β, IL-6, and TNF-α) mRNAs were significantly increased under MDP stimulation (P<0.001).  Moreover, SLC15A4-Knockout (SLC15A4-KO) cells were obtained through lentivirus packaging, transfection, screening, and cell monoclonal culture.  In order to gain further insight into the potential function of SLC15A4, we utilized transcriptome data, which revealed a change in the genes between WT-BRECs and SLC15A4-KO.  Five down-regulated pro-inflammatory genes and 13 down-regulated chemokine genes related to the inflammatory response were identified.  Meanwhile, the down-regulated genes were mostly enriched in the nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways.  The results of RT-qPCR also verified these detected changes.  To further determine the mechanism of how WT and SLC15A4-KO BRECs are involved in inflammatory responses, we investigated the inflammatory responses of cells exposed to MDP.  WT-BRECs and SLC15A4-KO were treated with a culture medium containing 10 μg mL^–1 MDP, in comparison to a control without MDP.  Our results show that SLC15A4-KO BRECs had reduced the expression of genes (IL-6, TNF-α, CXCL2, CXCL3, CXCL9, and CCL2) and proteins (p-p65 and p-p44/42) from the MDP-mediated inflammatory response compared to WT-BRECs (P<0.05).  In this experiment, CRISPR-Cas9 was used to KO the di/tripeptide transporter SLC15A4, and its role was confirmed via the MDP-induced inflammatory response in BRECs.  This work will provide a theoretical basis for studying the pro-inflammatory mechanism of MDP and its application in the prevention and treatment of subacute rumen acidosis in dairy cows.