Tumor necrosis factor-alpha (TNF-a) is a pleiotropic inflammatory cytokine. Interstitial fibrosis is prominent in the Dahl salt-sensitive (SS) rat, including in the outer medullary region where thick ascending limbs (TALs) are abundant. We found previously that members of the miR-29 microRNA family, which have potent anti-fibrotic effects, were expressed at a lower level in the kidneys of SS rats. In the present study, we examined the role of TNF-a in the regulation of miR-29 and fibrotic genes in the kidney. RNA-seq analysis showed that SS rats fed a high salt diet had higher expression levels of TNF-a and TNFRSF1a which is one of its main receptors compared with a 0.4% salt diet group. Rat medullary thick ascending limb (mTAL) cells were treated with TNF-a at 25ng/ml for 24h or 48h. At 24h, TNF-a significantly decreased the abundance of miR-29b (to 57% ± 9% of control, n=14, p<0.05) and miR-29c (to 74% ± 6% of control, p<0.01) and significantly increased alpha-smooth muscle actin (a-SMA) by more than two fold and up-regulated extracellular matrix genes such as Col4a1 (to 161% ± 25% of control, n=10, p<0.05) and Col4a2 (to 172%± 18% of control, p<0.01). At 48h, TNF-a significantly suppressed miR-29a and again up-regulated a-SMA and Col4a2. Similarly, TNF-a suppressed miR-29 in primary cultures of human renal epithelial cells, an effect that was attenuated by inhibition of NF-kB. This study shows that TNF-a can suppress miR-29 and up-regulate fibrotic genes in renal epithelial cells including mTAL cells, which may play an important role in the development of renal tubulointerstitial injury in SS rats.