The objective of this study was to target rutin, in a more solubilized form, to the colon aiming at treatment of colon carcinoma. pH sensitive nanospheres were prepared by the nanoprecipitation technique employing Eudragit S100. Different drug: polymer ratios as well as different concentrations of the stabilizer Poloxamer-188 were used. The developed rutin nanospheres exhibited entrapment efficiency ranging from 94.19% to 98.1%, with a zeta potential values <-20 mV. They were spherical in shape and their sizes were in the nanometric dimensions. The in vitro release study of nanospheres formulations revealed enhancement of aqueous solubility of rutin and indicated drug targeting to the colon. The selected formulations were stable after storage for 6 months at ambient room and refrigeration temperatures. In vitro cytotoxic study was conducted on human colon cancer (HCT-116) as well as normal human fibroblasts (BHK) cell lines, employing Sulphorhodamine-B assay. Rutin nanospheres showed significantly (P = .001) higher area under inhibition percentage curve, when compared to free drug, revealing more than 2-fold increase in rutin cytotoxic activity. These results reveal that Eudragit S100 nanospheres could be a potential drug delivery system to the colon with enhanced solubility and hence improved the cytotoxic activity of rutin.