Objective To investigate the changes of expression of 5-hydroxy tryptamine (5-HT), substance P (SP) in rat colon during heroin withdrawal, methadone detoxication and heroin relapse period. Methods Thirty-five adult male SD rats were randomly divided into three groups: normal control group (NCG), saline control group (SCG) and experimental group (EG). Experimental group divided into: heroin withdrawal group(HWG), methadone detoxication group(MDG) and heroin relapse group(HRG).The colon tissues of rats in five groups were excised respectively. Immunohistochemical SABC method and image analysis were used in the research. Results Compared with NCG and SCG, the mean grey degree of 5-HT, SP immunoreactive cells decreased, the number of 5-HT,SP immunoreactive cells increased obviously in HWG and HRG(P<0.05). Compared with NCG and SCG, there were no statistical significances on the mean grey degree and cells counting of 5-HT and SP immunoreactive cells (P>0.05) in MDG. Conclusion The colonic secretion of 5-HT and SP increases during heroin withdrawal and relapse. After treatment with methadone detoxication,the expression of 5-HT and SP is close to normal. The result suggests that the colonic secretion of 5-HT and SP participate in the process of body adjusting during heroin withdrawal, methadone detoxication and heroin relapse period.