To discuss the effect of hydrophobic groups of a polymer on the structural properties and function of polymer nanoparticles (NPs), we grafted chenodeoxycholic acid (CDCA) with pullulan (PU) to form hydrophobically modified PU (PUC). Three PUC polymers, namely, PUC-1, PUC-2, and PUC-3, with different degrees of substitution were designed by changing the feed ratio of CDCA and PU. 1H-NMR spectra showed that the PUC polymer was successfully synthesized, and the degrees of hydrophobic substitution for PUC-1, PUC-2, and PUC-3 were calculated to be 10.66%, 13.92%, and 16.94%, respectively. The PUC NPs were prepared by the dialysis method and were shown to be uniformly spherical by transmission electron microscopy (TEM). The average sizes were about (220±10) nm, (203±7) nm, and (163±6) nm under dynamic light scattering (DLS) for PUC-1 NPs, PUC-2 NPs, and PUC-3 NPs, respectively. Drug release experiments showed that the three PUC/DOX NPs exhibited good sustained release. At 48 h, the IC50 of doxorubicin in inhibiting colon cancer HCT116 cells was 0.0904 μg/mL. A cell study showed that PUC-3/DOX NPs had the highest uptake efficiency by HCT116 cells with the most cytotoxicity and inhibited the migration of HCT116 cells with the highest efficiency. The structural properties and function of polymer NPs were closely related to the hydrophobic groups in the polymer, and NPs with higher hydrophobicity showed a smaller size, higher drug capacity, and greater cell efficiency.