Little is known about the time-course of aging on interstitial cells of Cajal (ICC) of colon. The aim of this study was to investigate the change of morphology, ICC and neuronal nitric oxide synthase (nNOS) immunoreactive cells in the aged rat. The proximal colon of 344 Fischer rats at four different ages (6, 31, 74 weeks, and 2 years) were studied. The immunoreactivity of c-Kit, nNOS, anti-protein gene product 9.5(PGP 9.5) and synaptophysin were counted after immunohistochemistry. The c-kit, SCF (stem cell factor; ligand of Kit) and nNOS mRNA were measured by real-time PCR. c-Kit and nNOS protein were assessed by Western blot. Isovolumetric contractile force measurement and electrical field stimulation (EFS) were conducted. The area of intramuscular fat deposition significantly increased with age after 31 weeks. c-Kit immunoreactive ICC and nNOS immunoreactive neurons and nerve fibers significantly declined with age. mRNA and protein expression of c-kit and nNOS decreased with aging. The functional study showed that the spontaneous contractility was decreased in aged rat, whereas EFS responses in the presence of atropine and L-NG-Nitroarginine methyl ester were increased in aged rat. In conclusion, the decrease of proportion of proper smooth muscle, the density of ICC and nNOS immunoreactive neuronal fibers and the number of nNOS immunoreactive neurons during the aging process may explain the aging-associated colonic dysmotility.