Objective To investigate the effect of the heroin addicts on substance P (SP), neuropeptide Y (NPY) immunoreactive cells in the ventral tegmental area (VTA) and nucleus accumbens (NAc) neurons of the rat brain, and to explore the central mechanisms of heroin dependence and to provide experimental and theoretical basis. BR>Methods Fifty-five adult male SD rats were randomly divided into heroin dependence group (HDG), saline control group (SCG) and normal control group (NCG). Rats were chronically treated by injection of heroin for up to 38 days, and brain tissues were excised on the 10th, 17th, 24th, 31st, and 38th day after models were set up. Immunohistochemical SABC method, and image analysis were performed to test the expression of SP and NPY in the VTA and NAc regions. Results SP immunoreactive cells and NPY immunoreactive cells expressed less SP and NPY in the VTA and NAc regions from heroin dependence rats than that from saline control and normal control rats at different time points. Immunohistochemical analysis showed that the grey value was found higher in heroin dependence rats than that in controls (EM>P/EM> 0.05).Conclusion The secretion of SP and NPY is inhibited during heroin dependence and it may result in the inhibition of endogenous opioid peptides, suggesting that SP and NPY may be the key molecules during dru