We describe a new cell-penetrating protein, B1, capable of delivering conjugated proteins and nucleic acids into mammalian cells. B1 is a 244-amino-acid product of a single-base frameshift in the gene encoding enhanced green fluorescent protein (eGFP). The molecule has a net positive charge of 43 and a very high charge-to-mass ratio of 1.5. eGFP-fused B1 potently penetrates both adherent and suspension cells with >80% of cells taking up the protein when exposed to concentrations as low as 1 μM. The protein was found to cluster in the paranuclear region of TZM-bl cells. Most importantly, we show that B1 not only facilitates cellular uptake but allows biomolecular cargo to reach sites of biological relevance. For example, baby hamster kidney cells underwent DNA recombination when exposed to B1-tagged Cre recombinase at protein concentrations as low as 2.5 μM, indicating potent nuclear delivery of functional protein cargos. Additionally, B1 delivers noncovalently conjugated RNA and DNA across the cell membrane to cytosolic and nuclear sites accessible to the cellular translation and transcription machinery, as gauged by detection of encoded reporter functions, with efficiency comparable to commercially available cationic lipid reagents. B1 appears to utilize cell-surface glycans and multiple competing endocytic pathways to enter and traffic through cells. These studies provide both a new tool for intracellular delivery of biomolecules and insights that could aid in the design of more effective cell penetrating proteins.