An unmet need in nanomedicine is to prepare biocompatible and renal clearable nanoparticles by controlling the diameter, composition and surface properties of the nanoparticles. This paper reports cellulose nanofiber templated synthesis of ultra-small bismuth nanoparticles, and their uses in enhanced X-ray radiation therapy. The interstitial spaces between adjacent fibers are the adsorption sites of bismuth ions and also stabilize nanoparticles generated by chemical reduction. The sizes of nanoparticles are tailored in the 2-10 nm range using cellulose nanofibers with various amounts of carboxyl groups. In vitro cytotoxicity, reactive oxygen species (ROS) and in vivo animal tests with tumor-bearing mice are studied in order to enhance X-ray radiation therapy using cellulose nanofiber-templated bismuth nanoparticles. Bismuth nanoparticles show strong X-ray attenuation ability, concentration-dependent cytotoxicity and high level production of ROS upon X-ray exposure, which is consistent with enhanced cellular damage and retarded growth of tumors in animals.