Hybrid mesoporous silica nanoparticles (MSNs), which were synthesized using the co-condensation method and engineered with unique redox-responsive gatekeepers, were developed for studying the glutathione-mediated controlled release. These hybrid nanoparticles constitute a mesoporous silica core that can accommodate the guests (i.e., drug, dye) and the PEG shell that can be connected with the core via disulfide-linker. Interestingly, the PEG shell can be selectively detached from the inner core at tumor-relevant glutathione (GSH) levels and facilitate the release of the encapsulated guests at a controlled manner. The structure of the resulting hybrid nanoparticles (MSNs-SS-mPEG) was comprehensively characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), powder X-ray diffraction (XRD), and nitrogen adsorption/desorption isotherms analysis. The disulfide-linked PEG chains anchored on MSNs could serve as efficient gatekeepers to control the on-off of the pores. Compared with no GSH, fluorescein dye as the model drug loaded into MSNs showed rapid release in 10 mM GSH, indicating the accelerated release after the opening of the pores regulated by GSH. Confocal microscopy images showed a clear evidence of the dye-loaded MSNs-SS-mPEG nanoparticles endocytosis into MCF-7 cells and releasing guest molecules from the pore inside cells. Moreover, in vitro cell viability test using MTT assay indicated that MSNs-SS-mPEG nanoparticles had no obvious cytotoxicity. These results indicate that MSNs-SS-mPEG nanoparticles can be used in the biomedical field.