Enteropathogenic Escherichia coli (EPEC), a food borne human pathogen, is responsible for infantile diarrhea, especially in developing countries. The pathophysiology of EPEC-induced diarrhea, however, is not completely understood. Our recent studies showed modulation of Na⁺/H⁺ and Cl^-/HCO₃ ^- exchange activities in Caco-2 cells in response to EPEC infection. We hypothesized that intestinal short chain fatty acids (SCFA) absorption mediated by monocarboxylate transporter 1 (MCT1) might also be altered by EPEC infection. The aim of the current studies was to examine the effect of EPEC infection on butyrate uptake. Caco-2 cells were infected with wild type EPEC, various mutant strains, or non-pathogenic E. coli HS4 and ¹⁴C butyrate uptake was determined. EPEC, but not non-pathogenic E. coli significantly decreased butyrate uptake. Infection of cells with strains harboring mutations in escN, which encodes a putative ATPase for the EPEC type III secretion system (TTSS), or in the espA, espB or espD, genes encoding structural components of the TTSS, had no effect on butyrate uptake, indicating the TTSS dependence. On the other hand, strains with mutations in the effector protein genes espF, espG, espH and map inhibited butyrate uptake, similar to the wild type EPEC. Surface expression of MCT1 decreased considerably following EPEC, but not non-pathogenic E. coli infection. In conclusion, our studies demonstrate inhibition of MCT1-mediated butyrate uptake in Caco-2 cells in response to EPEC infection. This inhibition was dependent on a functional TTSS and the structural proteins EspA, B and D of the translocation apparatus.