Dietary substances can manipulate the expression of endogenous host defense peptides (HDPs), which may provide a promising strategy for disease control and prevention, especially for antibiotic-resistant infections. We hypothesized that butyrate can induce HDP expression, which likely contributes to the elimination of Escherichia coli O157:H7 in the intestine and reduces the severity of inflammation caused by E. coli O157:H7 challenge. Piglets treated with or without butyrate (2 g/kg of diet) 2 d before E. coli O157:H7 challenge was employed to investigate the relationship among porcine HDP expression, status of inflammation and E. coli O157:H7 load in feces. The effects of butyrate on HDP gene expression, antibacterial activity of 3D4/2 macrophages and IPEC-1 cells, and phagocytic capacity of 3D4/2 and polymorphonuclear (PMN) leukocytes in vitro were also examined. Data were represented as mean ± SEM. One-way ANOVA or Student’s t-test was used to compare differences between treatment groups, P < 0.05 was considered statistically significant. Results showed that butyrate treatment (1) alleviated clinical symptoms of E. coli O157:H7-induced hemolytic uremic syndrome and severity of intestine inflammation, (2) significantly reduced E. coli O157:H7 load in feces 24 h after the last inoculation (P < 0.01), (3) significantly upregulated multiple HDPs, such as pBD2, pBD3, PMAP23, PR-39, and PG1–5 (P < 0.05), but not all HDPs, in vitro and in vivo, and (4) enhanced the antibacterial activity of macrophage 3D4/2 and IPEC-1 and the phagocytic capacity of 3D4/2 and PMN cells in vitro. Our findings indicate that butyrate enhances disease resistance, promotes clearance of E. coli O157:H7, and alleviates clinical symptoms of hemolytic uremic syndrome and inflammation partially by affecting endogenous HDP expression.