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Although the delivery of biologically functional protein(s) into mammalian cells could be of tremendous value to biomedical research, the development of such technology has been hindered by the lack of a safe and effective delivery method. Here, we present a simple, efficient, and versatile gold nanoparticle–DNA aptamer conjugate (AuNP–Apt)-based system, with nanoblock-like properties, that allows any recombinant protein to be loaded without additional modifications and delivered into mammalian living systems. AuNP–Apt-based protein delivery system was able to deliver various proteins into variety of cell types in vitro without showing cytotoxicity. This AuNP–Apt system was also effective for the local and systemic targeted delivery of proteins in vivo. A local injection of the AuNP–Apt loaded with the apoptosis-inducing BIM protein efficiently inhibited the growth of xenograft tumors in mice. Furthermore, an intravenous injection of AuNP–Apt loaded with both epidermal growth factor (EGF) and BIM resulted in the targeted delivery of BIM into a xenograft tumor derived from EGF receptor-overexpressing cancer cells with no detectable systemic toxicity. Our findings show that this system can serve as an innovative platform for the development of protein-based biomedical applications.Schematic diagram of the generation of functionalized AuNPs conjugated with a His-DNA aptamer and His-tagged protein.