BACKGROUND: Diabetic cystopathy (DCP) is linked to bladder nerve conduction disorders, with diabetes-induced neuropathy impairing nerve signal transmission and causing bladder dysfunction. Myosin 5a, vital for neuronal transport, has been linked to neurological disorders, though its role in DCP remains unclear. The objective of this study was to investigate whether Myosin 5a plays a potential regulatory role in Diabetic Cystopathy. METHODS: Bladder strips from diabetic rats were use to assess heightened responsiveness to external stimuli. Urodynamic assessments were conducted to track the progression of bladder voiding dysfunction over time, following streptozotocin (STZ) injection. Single-cell RNA-Seq mining was employed to identify associations between Myosin 5a and bladder overactivity. Cellular and tissue analyses were performed to determine the co-localization of Myosin 5a with neurotransmitter-related proteins. The impact of Myosin 5a knockdown on ChAT and SP expression in bladder neurons was also evaluated. Additionally, Myosin 5a-deficient DBA mice were studied for voiding function and sensitivity to stimuli. Student's t-test (two-tailed) or Mann-Whitney's U test analysis of variance was used to analyze the difference between groups. RESULTS: Bladder strips from diabetic rats exhibit increased responsiveness to external stimuli, with urodynamic assessments showing a progressive decline in bladder function, culminating in overactivity by the fourth week post-STZ injection. Co-localization of Myosin 5a with neurotransmitter-related proteins was observed, and the knockdown of Myosin 5a in bladder neurons led to a significant reduction in ChAT and SP expression. Myosin 5a-deficient DBA mice exhibited abnormal voiding function and reduced sensitivity to stimuli, along with significant downregulation of SLC17A9. Single-cell RNA-Seq analysis revealed a significant link between Myosin 5a and bladder overactivity, with Myosin 5a expression escalating in tandem with the severity of bladder dysfunction. CONCLUSIONS: Myosin 5a's dysregulation in diabetic rats may worsen bladder overactivity, suggesting its potential as a therapeutic target for diabetic OAB.