Inappropriate Na⁺ reabsorption by thick ascending limbs (THALs) induces hypertension. NO produced by NO synthase type 3 (NOS3) inhibits NaCl reabsorption by THALs. Tumor necrosis factor α (TNF-α) decreases NOS3 expression in endothelial cells and contributes to increases in blood pressure. However, the effects of TNF-α on THAL NOS3 and the signaling cascade are unknown. TNF-α activates several signaling pathways, including Rho/Rho kinase (ROCK), which is known to reduce NOS3 expression in endothelial cells. Therefore, we hypothesized that TNF-α decreases NOS3 expression via Rho/ROCK in rat THAL primary cultures. THAL cells were incubated with either vehicle or 1 nmol/L of TNF-α for 24 hours, and NOS3 expression was measured by Western blot. TNF-α decreased NOS3 expression by 51±6% (P<0.002) and blunted stimulus-induced NO production. A 10-minute treatment with TNF-α stimulated RhoA activity by 60±23% (P<0.04). Inhibition of Rho GTPase with 0.05 μg/mL of C3 exoenzyme blocked TNF-α–induced reductions in NOS3 expression by 30±8% (P<0.02). Inhibition of ROCK with 10 μmol/L of H-1152 blocked TNF-α–induced decreases in NOS3 expression by 66±15% (P<0.001). Simultaneous inhibition of Rho and ROCK had no additive effect. Myosin light chain kinase, NO, protein kinase C, mitogen-activated kinase kinase, c-Jun amino terminal kinases, and Rac-1 were also not involved in TNF-α–induced decreases in NOS3 expression. We conclude that TNF-α decreases NOS3 expression primarily via Rho/ROCK in rat THALs. These data suggest that some of the beneficial effects of ROCK inhibitors in hypertension could be attributed to the mitigation of TNF-α–induced reduction in NOS3 expression.