Technical Abstract: We investigated the efficacy of feed supplemented with caprylic acid, a natural, 8-carbon fatty acid for reducing Salmonella Enteritidis colonization in commercial broiler chickens. In two separate 3- and 6-wk trials, day-old straight run (N=70 per trial), chicks were assigned to 5 treatment groups (n=14/group): a positive control (challenged with S. Enteritidis, no caprylic acid) and 2 identical challenged groups each for 0.7 % or 1% caprylic acid. For 3-wk trial, on d 5, birds were challenged with S. Enteritidis 4-strain mix by crop gavage and after 5th d post challenge (PC), 2 birds from each group were sacrificed to ensure S. Enteritidis colonization. Caprylic acid supplementation was carried out from d 15 for 5 d followed by sacrifice for tissue samples. For 6-wk trial, on d 25, birds were challenged and confirmed for S. Enteritidis. The birds were sacrificed for tissue samples after caprylic acid supplementation for the last 5 d. Caprylic acid at 0.7 and 1% decreased S. Enteritidis populations significantly in cecum, small intestine, cloaca, liver, spleen and crop of caprylic acid -treated birds. Further, to elucidate a potential antibacterial mechanism of action of caprylic acid, we investigated if caprylic acid could reduce S. Enteritidis invasion of an avian epithelial cell line, and expression of invasion genes hilA and hilD. The cell culture data showed that caprylic acid reduced invasion capabilities of all S. Enteritidis strains significantly by 60-90%. Gene expression studies indicated that caprylic acid significantly down-regulated Salmonella invasion genes hilA and hilD. These results suggest that supplementation of caprylic acid through feed could significantly reduce S. Enteritidis colonization in broiler chicken, and potentially reduces the pathogen’s ability to invade intestinal epithelial cells by down-regulating hilA and hilD.