Feline idiopathic cystitis (FIC) is one of the most common urinary tract disorders in domestic cats. As stress is suggested to play a key role in the pathogenesis of FIC, the effects of norepinephrine (NE) as a stress mediator were investigated on a novel feline primary uroepithelial cell culture, serving as an in vitro model of the disease. The uroepithelial cells gained from the mucosa of the bladder of a euthanized cat were cultured for 6 days and were acutely exposed to NE (10, 100, and 1000 µM) for 1 h. NE increased the metabolic activity of the cultured cells and elevated the extracellular concentrations of the pro-inflammatory mediators interleukin-6 (IL-6) and stromal cell derived factor 1 (SDF-1), confirming that NE can trigger an inflammatory response in the uroepithelium. Cellular protein carbonyl levels were increased by NE exposure, while malondialdehyde and glucose regulated protein 78 concentrations remained unchanged, indicating that NE may provoke the oxidative damage of proteins without inducing lipid peroxidation or endoplasmic reticulum stress. Further, it can be strongly suggested that an acute NE challenge might diminish the barrier function of uroepithelial cells, as reflected by the decreased glycosaminoglycan concentration, claudin-4 protein expression, and reduced TER values of the NE-treated cell cultures. Based on these results, short-term NE exposure mimicking acute stress can provoke an inflammatory response and decrease the barrier integrity of cultured feline uroepithelial cells. Hence, it is highly expected that stress-associated NE release may play an important mediatory role in the pathogenesis of FIC.