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Objectives: Nitric oxide is a neurotransmitter that is related to overactive bladder. This study was undertaken to evaluate the expression of nitric oxide synthases (NOS) in relation to bladder function after the removal of the bladder outlet obstruction in rats. Materials and Methods: 50 rats were divided into a control group (n = 10) and an experimental group (n = 40). The bladders in the experimental group were partially obstructed for 3 weeks. 3 weeks after removal, cystometrograms (CMG) were performed. On the basis of CMG results, the experimental group was subdivided into ‘normalized’ and ‘overactive’ groups. Expression of neuronal, inducible, and endothelial NOS mRNA in bladders of each group was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR). Results: On the CMG, the contraction interval of the overactive group was markedly decreased. RT-PCR results showed significantly decreased expression of nNOS and iNOS mRNA in the normalized group compared to the control group, and increased nNOS levels in the overactive group, compared to the normalized group (p < 0.05). A marked increase in eNOS mRNA expression was evident in the normalized and overactive groups compared to the control group (p < 0.05). Conclusions: Our results clearly show increases in nNOS and eNOS expression according to bladder function, which may be related to a persistently overactive bladder or irritative symptoms, following the removal of a bladder outlet obstruction.