Background: The female urinary bladder is a target organ for estrogen. Reductions in circulating estrogen have been associated with urothelial and vaginal atrophy and bladder disorders including incontinence and increased incidence of bladder infections. We determined the effect of short-term ovariectomy on sex hormones, bladder blood flow, and tissue oxygenation in the rabbit model. Materials and Methods: Female New Zealand White rabbits were ovariectomized and evaluated on 1, 3, and 7 days after ovariectomy. Tissue oxygenation (pO2) and blood flow were measured with oxylab system of real time measurements. Serum estrogen and progesterone were determined at sacrifice. Tissue hypoxia was localized histologically using Hypoxyprobe-1 immunohistochemistry. Results: Short-term ovariectomy caused rapid decreases in serum estrogen and progesterone, significant decreases in urothelial oxygenation and blood flow. No significant decreases in blood flow or oxygenation were noted for the detrusor smooth muscle. Immunohistochemistry confirmed the presence of urothelial hypoxia at all times after ovariectomy. Bladder muscle did not demonstrate significant levels of hypoxia. Conclusion: The bladder urothelium is extremely sensitive to short-term ovariectomy, with significant urothelial hypoxia seen by post-ovariectomy day 1. Urothelial hypoxia may play a significant role in pelvic pain syndromes, incontinence, and increased susceptibility to bladder infection.