The mixture of 2'-O-(4-carboxybutyryl)-5-fluorouridine (2'-glu-FUR) and 3'-O-(4-carboxybutyryl)-5-fluorouridine (3'-glu-FUR), named (glu-FUR(I)), and 5'-O-(4-carboxybutyryl)-5-fluorouridine (5'-glu-FUR), named glu-FUR(II), were easily obtained from the reaction of 5-fluorouridine (FUR) with glutaric anhydride. In addition, the chitosan-glu-FUR(I) conjugate and the chitosan-glu-FUR(II) conjugate were prepared. The obtained compounds were investigated regarding their in vitro characteristics. Equilibrium between 2'-glu-FUR and 3'-glu-FUR was suggested to be attained quickly in a 1/15 M phosphate buffer of pH 7.4. Glu-FUR(II) was found to be introduced into chitosan more easily than glu-FUR(I). For every compound, chemical hydrolysis was accelerated at weakly basic pH and a gradual regeneration of FUR was observed at physiological pH.