Background & Aims: Internal anal sphincter (IAS) tone plays an important role in rectoanal incontinence (RI). The IAS tone may be compromised during aging leading to RI in certain patients. Herein, we examined the influence of oxidative stress in the aging-associated decrease in the IAS tone (AADI). Methods: Using two different age groups of adult (4- 6 months (Ad)) vs. aging (24 to 30 months (Ag)) rats, we determined the effect of oxidative stress on IAS tone, and the regulatory RhoA/ROCK signal transduction cascade. We determined the effect of an oxidative stress inducer LY83583 (that produces superoxide anions or O₂^-), on the basal and stimulated IAS tone, before and after O₂^- scavenger superoxide dismutase (SOD) using intact smooth muscle (SM) strips and SMCs. Results: Data showed that AADI was associated with decrease in RhoA/ROCK expression at the transcriptional and translational levels. Oxidative stress with LY83583-mediated decrease in the IAS tone and relaxation of the IAS SMCs were associated with the decrease in RhoA/ROCK signal transduction, reversible by SOD. In addition, LY83583 caused a significant decrease in the IAS contraction produced by the RhoA activator, and a known RhoA/ROCK agonist U46619, also reversible by SOD. The inhibitory effects of LY83583 and ROCK inhibitor Y27632 on U46619-induced increase in the IAS tone were similar. Conclusion: An increase in oxidative stress plays an important role in the AADI in the elderly, and may be one of the underlying mechanisms for the RI in certain aging patients.