Hyaluronic acid (HA) coupled chitosan nanoparticles (HACTNP) bearing 5-flurouracil (5FU) were prepared, by ionotropic gelation method, for the effective delivery of drug to the colon tumors. HACTNP appeared to be spherical in shape and mean size was found to be around 150+/-3.4nm with low polydispersity index. The in vitro drug release was investigated using USP dissolution test (paddle type) apparatus in different simulated GIT fluids. The biocompatibility of NPs formulations were evaluated for in vitro cytotoxicity by MTT assay using HT-29 cell lines and cell uptake was assessed by fluorescent microscopy. Cellular uptake of HACTNP was determined by incorporating calcein as a fluorescent marker. The cellular uptake of fluorescent HACTNP was clearly evidenced by fluorescence microscopy. HACTNP nanoparticles showed significant higher uptake by cancer cells as compared to uncoupled nanoparticles and the uptake of HA coupled CTNPs by HT-29 colon cancer cells were observed to be 7.9 times more as compared to uncoupled CTNPs at the end of 4h. The cytotoxicity of 5FU incorporated in HACTNP was higher compared to the conventional 5FU solution, even at the lower concentrations. 5FU in HACTNP was about 2.60-folds more effective than free 5FU on HT-29 cells.