SLC26A3 (Down-regulated in adenoma, DRA) is a Cl^-/HCO₃^- exchanger involved in electroneutral NaCl absorption in the mammalian intestine. Altered DRA expression levels are associated with infectious and inflammatory diarrheal diseases. Therefore, it is critical to understand the regulation of DRA expression. MicroRNAs (miRNAs) are endogenous, small RNAs that regulate protein expression via blocking the translation and/or promoting mRNA degradation. In order to investigate potential modulation of DRA expression by microRNA, five different in silico algorithms were used to predict the miRNAs that target DRA. Of these miRNAs, miR-494 was shown to have a highly conserved putative binding site in the DRA 3'untranslated region (3'UTR) compared to other DRA-targeting miRNAs in vertebrates. Transfection with pmirGLO dual luciferase vector containing DRA 3'UTR (pmirGLO-3'UTR DRA) resulted in a significant decrease in relative luciferase activity as compared to empty vector. Co-transfection of the DRA 3'UTR luciferase vector with a miR-494 mimic further decreased luciferase activity as compared to cells transfected with negative control. The transfection of a miR-494 mimic into Caco2 and T-84 cells significantly increased the expression of miR-494 and concomitantly decreased the DRA protein expression. Mutation of the seed sequences for miR-494 in 3'UTR of DRA abrogated the effect of miR-494 on 3'UTR. These data demonstrate a novel regulatory mechanism of DRA expression via miR-494 and indicate that targeting this microRNA may serve to be a potential therapeutic strategy for diarrheal diseases.