This is the first study to determine the precise cellular localization of monocarboxylate transporter 4 (MCT4), along with its co-existence with its chaperone, CD147 in the ruminant gastrointestinal tract. Quantitative Western blot analysis demonstrated that the abundance of MCT4 protein was in the order of forestomach > large intestine > abomasum >or= small intestine. Immunohistochemistry and immunofluorescence confocal laser microscopy showed that MCT4 in the forestomach was confined to the cell membranes of strata corneum and granulosum, while diffuse cytoplasmic staining for MCT4 was visualized in strata spinosum and basale. In the epithelium cells lining the abomasum, MCT4 immunoreactive positivities were predominantly localized on the basolateral membranes. In the small intestine, MCT4 was localized at the brush borders and the basolateral membranes of the epithelial cells lining the villi, however it was mostly found on the apical membranes of the crypt cells. In the large intestine, the immunoreactivity for MCT4 differed between the surface epithelium and the crypts; in the surface epithelium, MCT4 was mainly localized at the apical membranes, whereas in the crypts it was predominantly expressed on the basolateral membranes of the lining epithelial cells. MCT4 was remarkably co-existed with CD147 along the bovine gastrointestinal tract. Our results suggest that MCT4 can play an important role in the transport of SCFA. The study also explored the potential functional collaboration between MCT1 and MCT4 and provided new insights into the mechanisms that mediate the transport of SCFA and other monocarboxylates in the different segments of the ruminant gastrointestinal tract.