Short-chain fatty acids (SCFA) produced by colonic microbiota fermentation of dietary fiber regulate cell responses via G protein-coupled receptor 41 (GPR41) for non-ruminant animals. However, the regulation of cell responses by SCFA and GPR41 remain unknown in ruminant animals. Therefore, the objective of study is to investigate the functions of SCFA and GPR41 in inflammatory responses in bovine rumen epithelial cells (BREC). Bovine ruminal epithelium tissues from 3 young Holstein calves were obtained from the Experimental Farm of Yang Zhou University. The wild type (WT) BREC were cultured in DMEM medium for 24 h. Inflammatory responses were induced in WT BRECs and GPR41 knockdown (GPR41KD) BRECs with 20 mM SCFA (12 mM sodium acetate, 5 mM sodium propionate, and 3 mM sodium butyrate) for 24 h of incubations. Statistical analysis was performed by one-way ANOVA followed by the least significant difference (LSD) test for post-hoc correction for multiple comparisons of treatment means using the SPSS 16.0 software. P < 0.05 was considered significant. These results showed that the concentrations of 20 mM SCFA significantly enhanced IL1B, TNF, and CCL20, CXCL2, CXCL3, CXCL5, CXCL8, CXCL14 chemokines in WT BREC compared with WT BREC in the absence of 20 mM SCFA (P < 0.01). In comparison with WT BREC with 20 mM SCFA, the GPR41KD BREC with 20 mM SCFA significantly enhanced the proinflammatory cytokine of IL1B and TNF expression (P < 0.05), and reduced the expression of CCL20, CXCL2, CXCL3, CXCL5, CXCL8, CXCL14 chemokines, Occludin, and ZO1 (P < 0.05). Remarkably, GPR41KD BREC markedly decreased the peripheral blood mononuclear cells (PBMC) recruitment compared with WT BREC. The regulation of PBMC recruitment by GPR41 correlated well with immunological barrier protection. These findings revealed that SCFA regulate GPR41-mediated immune cell migration and thereby mediate protective immunity in BREC.