Polyethylene glycol (PEG)-immobilized quantum dot (QD) nanoparticles, which could be specifically dePEGylated in response to the presence of the matrix metalloprotease-2 (MMP-2) enzyme, were prepared. The degree of PEGylation (MW 3400) on the surface of 12 nm streptavidin-coated QDs was stoichiometrically controlled by varying the feed amount of a biotin-substrate-PEG conjugate, where the substrate contained an MMP-2 cleavable peptide sequence. A biotin-cell penetrating peptide (CPP) conjugate was also immobilized onto the surface of the PEGylated QD surface to enhance the cellular uptake after dePEGylation. It was found that more than nine PEG chains per single QD were required to effectively inhibit the cellular uptake of modified QD particles down to around 20%, as compared with that of QD without PEG chains. However, the treatment of MMP-2 enzyme in the medium resulted in a substantial enhancement in the extent of QD cellular uptake by dePEGylation with concomitant resurfacing of sterically hidden CPP moieties. This study analyzed the effects of surface PEGylation density and MMP-2 specific dePEGylation on the cellular uptake of CPP-QD nanoparticles in a quantitative manner.