MicroRNAs (miRNAs) play important roles in biological development and disease. Much less is known about their role in normal adult physiology. The proximal convoluted tubule (PCT) and the medullary thick ascending limb (mTAL) in the kidney consist of epithelial cells with different transport activities. We identified 55 possible miRNA-target pairs of which the miRNAs and their predicted target proteins, many of which are involved in epithelial transport, were inversely enriched in PCT and mTAL. Some miRNAs appeared to have synergistic effects on shared targets. miR-192 and its predicted target the β-1 subunit of Na+/K+-ATPase (Atp1b1), an enzyme providing the driving force for tubular transport, were inversely enriched in kidney regions. In mice, knockdown of miR-192 led to up-regulation of Atp1b1 protein. When mice were fed with a high-salt diet, knockdown of miR-192 blunted the adaptational increase of urine output. Interestingly, miR-192 appeared to target Atp1b1 through the 5′-, rather than 3′-untranslated region. The study suggests a novel physiological mechanism in which miR-192 suppresses Na+/K+-ATPase and contributes to renal handling of fluid balance. It supports an important role of miRNAs in determining cellular characteristics that may appear subtle yet are physiologically critical. © 2012 The Author(s).