The purpose of this study was to develop and evaluate the berberine hydrochloride (HB) - Eudragit S100 solid dispersion to prolong the drug release by expoiting its pH-sensitivity dissolution. Solid dispersion of berberine hydrochloride (HB) with Eudragit® S100 (S100) was prepared by a solvent evaporation method. Based on the evaluating dissolution in vitro, the ratio of HB and S100 was optimized to 1:4. The solid dispersion formation of HB-S100 was characterized by XRD, DSC, and results showed that berberine hydrochloride and Eudragit® S 100 was interacted to form a new complex in amorphous form. In-vitro cytotoxicity studies were evaluated in human colon cancer cells by MTT assay. The 50% inhibitory concentrations of HB for SW480, HCT116 and Caco-2 at 48 h were 43.10, 34.13 and 32.82 μM respectively. When formed dispersion with S100 the 50% inhibitory concentrations for SW480, HCT116 and Caco-2 decreased to 28.16, 22.06 and 15.39 μM. The results indicated that, compared with free HB, HB-S100 demonstrated lower IC50 values. Both HB and HB-S100 induced cells death in a dose- and time-dependent manner.