Multiple vaginal parities have been reported to be an important risk factor for stress urinary incontinence (SUI). Simulated birth trauma with single vaginal distention (VD) has been used to induce the SUI condition in animals; however, the effect of multiple simulated birth traumas on the urethral continence function has not been well characterized. Therefore, we examined the effects of multiple VDs on urethral functions in vivo and the changes in gene expressions of several molecules in the urethra using female SD rats, which were divided into 3 groups; sham, VD-1 (single VD), and VD-3 groups (3 times of VDs every 2 weeks). Two weeks after the final VD, leak point pressure (LPP) and urethral responses during sneezing were evaluated. Also, changes in mRNA levels of urethral molecules were quantified with RT-PCR. The VD-1 group did not show any change in LPP with only a tendency of decrease in amplitudes of the urethral responses during sneezing (A-URS); however, the VD-3 group showed a significant decrease in LPP and urethral responses such as baseline urethral pressure and A-URS accompanied with SUI episodes during sneezing. Nicotinic receptor subtypes and TGF-β1 were significantly increased in both VD-1 and VD-3 groups while TNFR-1, IL-6, collagens and MMP-9 were significantly increased only in the VD-3 group. These data indicate that rats with multiple simulated birth traumas exhibit the profound impairment of urethral continence function and that these functional changes are associated with those in cytokines, extracellular matrix molecules and nicotinic receptor subtypes in the urethra.