Dopamine-producing tyrosine hydroxylase (TH) neurons in the hypothalamic arcuate nucleus (ARC) have recently been shown to be involved in ghrelin signaling and body weight homeostasis. Here, we investigate the role of the intracellular regulator RhoA in hypothalamic TH neurons in response to peripheral hormones. Diet-induced obesity was found to be associated with increased phosphorylation of TH in ARC, indicating obesity-associated increased activity of ARC TH neurons. Mice, in which RhoA was specifically knocked out in TH neurons (TH-RhoA-/- mice), were more sensitive to the orexigenic effect of peripherally administered ghrelin and displayed an abolished response to the anorexigenic hormone leptin. When TH-RhoA-/- mice were challenged with a high-fat high-sucrose (HFHS) diet, they became hyperphagic and gained more body weight and fat mass compared to wildtype control mice. Importantly, lack of RhoA prevented development of ghrelin resistance, which is normally observed in wildtype mice after long-term HFHS diet feeding. Patch-clamp electrophysiological analysis demonstrated increased ghrelin-induced excitability of TH neurons in lean TH-RhoA-/- mice as compared to lean littermate control animals. Additionally, increased expression of the orexigenic hypothalamic neuropeptides AgRP and NPY was observed in TH-RhoA-/- mice. Overall, our data indicate that TH neurons in ARC are important for the regulation of body weight homeostasis and that RhoA is a central effector in these neurons and important for the development of obesity-induced ghrelin resistance. The obese phenotype of TH-RhoA-/- mice may be due to increased sensitivity to ghrelin and decreased sensitivity to leptin, resulting in increased food intake. This article is protected by copyright. All rights reserved.