This study reports the generation of protein-induced pluripotent stem cells (piPSCs) from murine embryonic fibroblasts using recombinant cell-penetrating reprogramming proteins. The authors designed and fused a poly-arginine (11R) protein transduction domain to the C-terminus of four reprogramming factors (Oct4, Sox2, Klf4, and c-Myc). These proteins were expressed in E. coli, solubilized, refolded, and purified. The purified proteins were shown to be stable and capable of entering cells within 6 hours at concentrations of 0.5–8 μg/ml. The piPSCs generated using this method were characterized for their long-term self-renewal, pluripotency markers, and global gene expression patterns, which were similar to those of conventional mouse embryonic stem cells (mESCs). In vitro and in vivo differentiation assays demonstrated that piPSCs could form embryoid bodies and differentiate into cells of all three germ layers, including endoderm, mesoderm, and ectoderm derivatives. Additionally, piPSCs efficiently incorporated into blastocysts and contributed to the germline in vivo. This study represents a significant advance in generating iPSCs by eliminating the need for genetic manipulation, offering a safer and more efficient method for iPSC generation.This study reports the generation of protein-induced pluripotent stem cells (piPSCs) from murine embryonic fibroblasts using recombinant cell-penetrating reprogramming proteins. The authors designed and fused a poly-arginine (11R) protein transduction domain to the C-terminus of four reprogramming factors (Oct4, Sox2, Klf4, and c-Myc). These proteins were expressed in E. coli, solubilized, refolded, and purified. The purified proteins were shown to be stable and capable of entering cells within 6 hours at concentrations of 0.5–8 μg/ml. The piPSCs generated using this method were characterized for their long-term self-renewal, pluripotency markers, and global gene expression patterns, which were similar to those of conventional mouse embryonic stem cells (mESCs). In vitro and in vivo differentiation assays demonstrated that piPSCs could form embryoid bodies and differentiate into cells of all three germ layers, including endoderm, mesoderm, and ectoderm derivatives. Additionally, piPSCs efficiently incorporated into blastocysts and contributed to the germline in vivo. This study represents a significant advance in generating iPSCs by eliminating the need for genetic manipulation, offering a safer and more efficient method for iPSC generation.