Antimicrobial resistance is a major concern to public health demanding effective alternative strategies to disease control and prevention. Modulation of endogenous host defense peptide (HDP) synthesis has emerged as a promising antibiotic alternative approach. This study investigated a potential synergy between sugars and butyrate in inducing HDP gene expression in chickens. Our results revealed that sugars differentially regulated HDP expression in both gene- and sugar-specific manners in chicken HD11 macrophage cells. Among eight mono- and disaccharides tested, all were potent inducers of avian β-defensin 9 () gene (<0.05), but only galactose, trehalose, and lactose obviously upregulated cathelicidin-B1 () gene expression. The expression ofgene, on the other hand, was minimally influenced by sugars. Moreover, all sugars exhibited a strong synergy with butyrate in enhancingexpression, while only galactose, trehalose, and lactose were synergistic with butyrate ininduction. No synergy ininduction was observed between sugars and butyrate. Although lactose augmented the expression of nearly all HDP genes, its synergy with butyrate was only seen with several, but not all, HDP genes. Mucin-2 gene was also synergistically induced by a combination of lactose and butyrate. Furthermore, lactose synergized with butyrate to induceexpression in chicken jejunal explants (<0.05). Mechanistically, hyper-acetylation of histones was observed in response to both butyrate and lactose, relative to individual compounds. Mitogen-activated protein kinase, NF-κB, and cyclic adenosine monophosphate signaling pathways were also found to be involved in butyrate- and lactose-mediated synergy ininduction. Collectively, a combination of butyrate and a sugar with both HDP-inducing and barrier protective activities holds the promise to be developed as an alternative to antibiotics for disease control and prevention.