Cytosolic protein delivery technique plays an important role in protein-based biotechnologies and therapeutics. However, the development of efficient nanocarriers for delivering cargo proteins into cytosols remains a continuing challenge due to the existence of multiple barriers. Here, we report an efficient strategy for the cytosolic delivery of native proteins by surface-engineered gold nanoparticles combined with hypertonicity treatment. Sub-10 nm gold nanoparticles stabilized by both cysteamine and 4-mercaptophenylboronic acid were used to complex cargo proteins via a combination of nitrogen-boronate coordination and ionic interactions. The yielding protein complexes with a size around 100 nm showed efficient endocytosis via micropinocytosis- and lipid raft-mediated pathways. Further the hypertonicity treatment of the transduced cells by glycerol, glucose, sucrose, and NaCl solutions efficiently facilitates the endosomal escape and the intracellular release of cargo proteins. By the proposed strategy, cargo proteins including bovine serum albumin, ovalbumin, green fluorescent protein, R-phycoerythrin, and horseradish peroxidase were successfully delivered into cell cytosol with maintained protein bioactivity. This study provides a feasible and efficient strategy for the intracellular protein delivery.