In this study, self-assembled nanoparticles based on amphiphilic xylan-stearic acid (Xyl-SA) conjugates have been developed for the efficient delivery of 5-fluorouracil (5-FU) in cancer therapy. The self-assembled behavior of Xyl-SA conjugates in aqueous medium was investigated using pyrene as fluorescent probe. To enhance the loading efficacy of 5-FU, the lipophilic 5-fluorouracil-stearic acid (5-FUSA) prodrug was synthesized and subsequently encapsulated into the hydrophobic core of Xyl-SA NPs. The obtained Xyl-SA/5-FUSA NPs had an appropriate size (~278 nm), high drug loading of 5-FUSA (~14.6 wt%) and high physiological stability. The interaction of the Xyl-SA/5-FUSA NPs with blood components was investigated by hemolysis study. The cell cytotoxic studies demonstrated that Xyl-SA/5-FUSA NPs induced higher cytotoxicity than free drugs against the Human colorectal cancer cells (HT-29, HCT-15). These results indicate that Xyl-SA/5-FUSA NPs can serve as a promising drug delivery system for the efficient delivery of 5-FU in cancer therapy.