Photo-labile protecting groups (PPG) allow for the selective activation of an originally caged moiety by light exposure at a specific wavelength. Incorporation of PPG in nanoparticles (NPs) enables precise spatiotemporal control over NPs surface properties. Thus, physicochemical and biological properties of NPs can be modified even after administration in a biological environment. In this study, this mechanism is used to control the cell uptake of NPs. To this end, polymeric core–shell NPs are used composed of poly(D, L-lactide-co-glycolide) and a poly(ethylene glycol)-b-poly(D, L-lactide) block copolymer, modified with positively charged cell-penetrating peptide (CPP). Surface charge of CPP-NPs (+23.50 mV), measured as zetapotential, is effectively diminished by the attachment of coumarin-derived PPG to CPP (+12.50 mV), resulting in reduced cell uptake. Upon light irradiation with light-emitting diode (λ = 365 nm) the PPG is cleaved, restoring the zetapotential (+24.67 mV) and triggering an enhanced cell uptake. This opens the door to trigger the cellular uptake of NPs that are intended to transport drugs to their target cells in the future.