In this in vivo study, we aimed to assess the radioprotective effect of amifostine on late normal tissue damage induced by gemcitabine concomitant with pelvic radiotherapy by histopathological and quantitative methods. Fifty-six male Wistar albino rats were randomly divided into seven experimental groups as follows: (I) gemcitabine, (II) radiation + gemcitabine, (III) radiation + gemcitabine + amifostine, (IV) radiation + amifostine, (V) sham radiation, (VI) amifostine, (VII) radiation. Irradiation was given to pelvic region with a dose of 25 Gy in 5 fractions. Amifostine was given for 30 min; gemcitabine was administered 24 h before the first fraction of radiotherapy. All animals were killed at the end of 4th month. Pathological examination was performed and the tissue collagen content was measured in bladder and rectal tissues. Fifty-one animals that were alive at the end of the follow-up period were analyzed. Thirty-five animals (68.6%) revealed grades I-III late effect in histopathological examination. We observed grade III colitis in 1 animal (radiation + gemcitabine) and bladder fibrosis in 4 animals (radiation and radiation + gemcitabine groups). There was no significant difference between any groups for bladder cystitis and fibrosis by Kruskal-Wallis method. Colitis was seen significantly lower in the radiation + gemcitabine + amifostine group (P = 0.0005). The collagen contents in the bladder and rectum of radiation and radiation + gemcitabine groups were markedly increased as compared to the sham group. This effect was reversed in the groups which received amifostine in addition to radiation and radiation + gemcitabine groups, but this difference was not significant. This study demonstrated that amifostine may have a beneficial effect in limiting rectal colitis from the radiosensitizing effect of gemcitabine.