Internal anal sphincter (IAS) tone plays an important role in rectoanal incontinence (RI). IAS tone may be compromised during aging, leading to RI in certain patients. We examined the influence of oxidative stress in the aging-associated decrease in IAS tone (AADI). Using adult (4–6 mo old) and aging (24–30 mo old) rats, we determined the effect of oxidative stress on IAS tone and the regulatory RhoA/ROCK signal transduction cascade. We determined the effect of the oxidative stress inducer LY83583, which produces superoxide anions (O₂^·−), on basal and stimulated IAS tone before and after treatment of intact smooth muscle strips and smooth muscle cells with the O₂^·− scavenger SOD. Our data showed that AADI was associated with a decrease in RhoA/ROCK expression at the transcriptional and translational levels. Oxidative stress with a LY83583-mediated decrease in IAS tone and relaxation of IAS smooth muscle cells was associated with a decrease in RhoA/ROCK signal transduction, which was reversible by SOD. In addition, LY83583 caused a significant decrease in IAS contraction produced by the RhoA activator and a known RhoA/ROCK agonist, U46619, that was also reversible by SOD. The inhibitory effects of LY83583 and the ROCK inhibitor Y27632 on the U46619-induced increase in IAS tone were similar. We conclude that an increase in oxidative stress plays an important role in AADI in the elderly and may be one of the underlying mechanisms of RI in certain aging patients.