Cytosolic protein delivery holds great potential for the development of protein-based biotechnologies and therapeutics. Currently, cytosolic protein delivery is mainly achieved with the assistance of various carriers. Herein, we present a universal and effective strategy for carrier-free cytosolic protein delivery via metabolic glycoengineering and bioorthogonal click reactions. Ac4ManNAz (AAM), an azido-modified N-acetylmannosamine analogue, was first employed to label tumor cell surfaces with abundant azido groups via glycometabolism. Then, proteins including RNase A, cytochrome C (Cyt C), and bovine serum albumin (BSA) were covalently modified with dibenzocyclooctyne (DBCO). Based on the highly efficient bioorthogonal click reactions between DBCO and azido, DBCO-modified proteins could be efficiently internalized by azido-labeled cancer cells. RNase A-DBCO could largely maintain its enzymatic activity and, thus, led to notable anti-tumor efficacy in HeLa and B16F10 cells in vitro and in B16F10 xenograft tumors in vivo. This study therefore provides a simple and powerful approach for carrier-free protein delivery and would have broad applicability in anti-tumor protein therapy.