The response of intestinal epithelial cells to short-chain fatty acids, which are increasingly used as food additives, was investigated. Human small intestinal epithelial cell model Caco-2 cells were exposed to formate, propionate and butyrate to assess their effect on cellular growth, metabolism, differentiation and protection against bacteria. The Caco-2 cells were entirely grown in the different short-chain fatty acids and respective growth patterns were determined. Differentiated cells were exposed to 0-20 mM short-chain fatty acids for 48 h and changes in DNA, RNA, (glyco)protein syntheses, sucrase isomaltase activity, transepithelial electrical resistance and protection against Salmonella enteritidis were measured. The short-chain fatty acids, altered linearly and differentially the growth pattern ranging from stimulation by formate to inhibition by butyrate. Formate inhibited cellular metabolism. Low concentrations of up to 5 mM propionate and 2 mM butyrate stimulated metabolism, while higher doses were inhibitory. Formate had no effect on sucrase isomaltase enzyme activity and transepithelial electrical resistance, whereas propionate and butyrate increased these markers of differentiation. Infection with S. enteritidis did not benefit from the short-chain fatty acid-induced transepithelial electrical resistance. It is concluded that formate, propionate and butyrate selectively and differentially modulate growth characteristics, cellular metabolism, sucrase isomaltase activity and transepithelial electrical resistance in a concentration- and carbon atom-related fashion. The short-chain fatty acid-induced transepithelial electrical resistance does not confer protection against S. enteritidis.