Effective delivery of proteins into the cytosol of mammalian cells would open the door to a wide range of applications. However, despite great efforts from numerous investigators, effective protein delivery in a clinical setting is yet to be accomplished. Herein we report a potentially general approach to engineering cell-permeable proteins by genetically grafting a short cell-penetrating peptide (CPP) to an exposed loop of a protein of interest. The grafted peptide is conformationally constrained, exhibiting enhanced proteolytic stability and cellular entry efficiency. Applying this technique to enhanced green fluorescent protein (EGFP), protein-tyrosine phosphatase 1B (PTP1B), and purine nucleoside phosphorylase (PNP) rendered all three proteins cell-permeable and biologically active in cellular assays. When added into growth medium at 0.5-5 μM concentrations, the engineered PTP1B dose-dependently reduced the phosphotyrosine levels of intracellular proteins, while the modified PNP corrected the metabolic deficiency of PNP-deficient mouse T lymphocytes, providing a potential enzyme replacement therapy for a rare genetic disease.