--- - |2- Abstract The in vivo application potential of viral‐based gene delivery approaches is hindered by a risk of insertional oncogenesis. Of the many delivery methods, cell‐penetrating peptides (CPP)‐based delivery has good biocompatibility and biodegradability. However, low efficiency is still the disadvantage of CPPs‐based nucleic acid transfection, and delivery efficiency may vary from different CPPs. Here, we describe Scp01‐b, as a new CPP, which can enter cultured cell lines and primary cultured cells examined by fluorescence microscopy and quantitative assay, the internalization process is a concentration, temperature, and incubation time‐dependent manner. Scp01‐b does not insert into the membrane directly and its uptake is mediated through endocytosis pathway. Moreover, Scp01‐b could mediate the uptake of plasmid DNA into the Caski and HSC‐T6 cells, and we noted that Scp01‐b‐mediated transfection efficiency was nearly the same with traditional liposome (TurboFectin)‐mediated transfection. These findings suggest that Scp01‐b can act as a useful tool for non‐viral‐based delivery in further application such as reprogramming and gene editing. - Journal of Cellular Physiology, EarlyView.