Polyglycerol dendrimers (PGDs), which exhibit a well-defined structure consisting of only glycerol units, were examined as a host molecule of 5-fluorouracil (5-Fu) used as a model anticancer drug. F-19- and H-1-NMR titrations and fluorescence measurements were performed to estimate the molecular interaction between PGDs and 5-Fu in a buffer. Results of the NMR titrations revealed that PGD of generation 3 (PGD-G3) encapsulated 5-Fu in the buffer, whereas PGD-G2 and G1 partially incorporated 5-Fu molecule into the space. Fluorescent spectra of 5-Fu in the presence of PGD-G3 indicated that the diketo (lactam) form of 5-Fu changed to the enol-keto (lactim) form of 5-Fu, suggesting attraction of the imine proton of 5-Fu by ether oxygen of PGD-G3. Therefore, the encapsulation state of 5-Fu in PGDs at molecular level was modulated by the well-defined branched structure depending on the generation of PGDs.