Introduction: The urinary retention problem reported in some patients using omeprazole and other proton pump inhibitors suggests that this drug has this effect by relaxing the bladder smooth muscle. Studies with omeprazole and other proton pump inhibitors show that these drugs cause relaxation in various tissue and organ smooth muscles, which also supports our hypothesis. However, there is no study in the literature about the possible effect and mechanism of action of omeprazole on bladder smooth muscle. The aim of this study is to investigate the possible effect and mechanism of action of omeprazole in isolated rat bladder detrusor muscle. Materials and Methods: In the presence of omeprazole (10-4, 5x10-4, 10-3 M), contraction responses induced by acetylcholine (10-6-10-3 M) and electrical field stimulation (2-64 Hz) were measured. Rac-1 pathway inhibitor NSC23799 (10-4 M) was used to investigate the mechanism of action of omeprazole. One-way analysis of variance (ANOVA) and Tukey-Kramer post-hoc test were used for statistical analysis. Results: Omeprazole (10-4, 5x10-4, 10-3 M) dose-dependently inhibited maximal contraction responses induced by acetylcholine and electrical field stimulation. When omeprazole (5x10-4 M) was applied in combination with NSC23766, it caused a significant increase in ACh and EFS-induced responses compared to the NSC23766 alone group. Conclusion: These results show that omeoprazole inhibits contractions in isolated rat detrusor muscle through blockade of Rac-1 pathway. Keywords: Detrusor Muscle, Omeprazole, Rac-1 Pathway, Rat