Fatty acid-binding protein (FABP) has high affinity for long-chain fatty acids and appears to participate in the metabolism and intracellular transport of lipids. Liver- and intestinal-type FABP (L-FABP and I-FABP, respectively) are expressed in the small intestine. However, in the gastrointestinal tract of ruminants, expression and localization of FABPs are unknown. In this study, we investigated the expression of I-FABP and L-FABP in the gastrointestinal tract of cattle. I- and L-FABP had higher messenger RNA (mRNA) and protein expression levels in the duodenum and jejunum relatively to other gastrointestinal regions in both calves and cows. Furthermore, L-FABP mRNA and protein expression were high in the colon. Both these protein types were confirmed to be in the cytosol of jejunal epithelial cells, where they were found in the villi rather than in the crypts. We concluded that duodenal and jejunal FABPs might be involved in the metabolism of fatty acids mainly in epithelial cells in cattle.