The aims of this study were to evaluate whether a diet supplemented with glyceryl butyrate could attenuate the immune-inflammatory response in piglets challenged with enterotoxigenic Escherichia coli (ETEC), and to explore the mechanisms of its regulation. Eighteen weaning piglets were assigned to three diets: basal diet (CON), antibiotics diet (ATB), and 0.5% glyceryl butyrate diet (GB group). Significantly lower concentrations of IL-1β, IL-6 and TNF-α in the jejunum and IL-6 in the ileum were observed in the GB group than that in the CON group (P < 0.05). Moreover, a decreasing trend of IL-1β (P = 0.075) and TNF-α (P = 0.070) was observed in the ileum in the GB group. Correspondingly, the GB group had significantly increased mRNA expression of porcine beta defensins (pBDs) in the jejunum (pBD1, pBD2, pBD114 and pBD129) and ileum (pBD2, pBD3, pBD114 and pBD129) (P < 0.05), and protein abundance of Claudin 1, Occludin, and ZO-1 in the jejunum and ileum (P < 0.05). Further research results showed that the improvement of beta defensins and tight junctions in the GB group was related to the decreased phosphorylation of the NFκB/MAPK pathway. In addition, the results of 16S rDNA sequencing showed that glycerol butyrate supplementation altered the ileal microbiota composition of piglets, increasing the relative abundance of Lactobacillus reuteri, Lactobacillus salivarius, and Lactobacillus agrilis. In summary, glyceryl butyrate attenuated the immune-inflammatory response in piglets challenged with ETEC by inhibiting the NF-κB/MAPK pathways and modulating the gut microbiota, and thus improved piglet intestinal health.