To develop a nano-sized drug delivery system for cancer therapy, amphiphilic hyaluronic acid conjugates were synthesized by chemical conjugation of hydrophobic 5β-cholanic acid to the backbone of hyaluronic acid (HA). The HA conjugates could form nano-sized self-aggregates under physiological conditions (PBS, pH = 7.4, 37 °C) via the hydrophobic interaction among 5β-cholanic acids. The HA nanoparticles were spherical in shape and their sizes were in the range of 350-400 nm, depending on the degree of substitution of 5β-cholanic acid. From a cellular experiment using Cy5.5-labeled HA nanoparticles, it was demonstrated that they are efficiently taken up by SCC7 cancer cells which over-express CD44, the receptor for HA. When the Cy5.5-labeled HA nanoparticles were systemically administrated into the tail vein of tumor-bearing mice, most of the nanoparticles were found in tumor and liver sites. In particular, the fluorescence intensity of nanoparticles at the tumor site was 4-fold higher than that of pure HA polymer, which was confirmed by a non-invasive near-infrared fluorescence imaging system. The high tumor targeting ability of HA nanoparticles might result from both their prolonged circulation in blood and high affinity to tumor cells. These results reveal the promising potential of HA nanoparticles as a stable and effective nano-sized drug delivery system for cancer treatment.