To develop a smart nanovalve on mesoporous silica nanoparticles (MSNs) for biomedical applications, a new type of peptide-functionalized MSN with a plug-gate nanovalve (PGN) was designed for targeted drug release in cancer cells. The outer shell of MSN was functionalized with K₈ peptide (octa-lysine sequence) by click chemistry, followed by reacting with citraconic anhydride via α,β-unsaturated bond to prepare negatively charged MSN–K₈(Cit). Subsequently, a cationic K₈(RGD)₂ peptide containing two Arg–Gly–Asp (RGD) sequences for targeting was introduced via electrostatic interaction to the negatively charged surface of MSN–K₈(Cit) to form PGN. It was found that, at pH 5.0 (simulating the endo/lysosomal environment), the surface charge of MSN–K₈(Cit) could convert from −41 mV to +19 mV due to the hydrolysis of the acid-labile amides in the acidic condition, implying the subsequent electrostatic repulsion to induce opening of the nanovalves and release of anticancer drug, DOX. According to the drug release studies, 79% of DOX was released within 48 h at pH 5.0, while much less DOX was released at pH 6.5 and 7.4. Furthermore, in vitro cellular experiments confirmed that the drug delivery system had enhanced cellular association and cell inhibition effect on α_vβ₃-positive U87 MG cancerous cells.