The cell-penetrating peptide (CPP) pep-1 is capable of introducing large proteins into different cell lines, maintaining their biological activity. Two possible mechanisms have been proposed to explain the entrance of other CPPs in cells, endosomal-dependent and independent types. In this work, we evaluated the molecular mechanisms of pep-1-mediated cellular uptake of beta-galactosidase (beta-Gal) from Escherichia coli in large unilamellar vesicles (LUV) and HeLa cells. Fluorescence spectroscopy was used to evaluate the translocation process in model systems (LUV). Immunofluorescence microscopy was used to study the translocation in HeLa cells. Enzymatic activity detection enabled us to monitor the internalization of beta-Gal into LUV and the functionality of the protein in the interior of HeLa cells. Beta-Gal translocated into LUV in a transmembrane potential-dependent manner. Likewise, the extent of beta-Gal incorporation was extensively decreased in depolarized cells. Furthermore, beta-Gal uptake efficiency and kinetics were temperature-independent, and beta-Gal did not colocalize with endosomes, lysosomes, or caveosomes. Therefore, beta-Gal translocation was not associated with the endosomal pathway. Although an excess of pep-1 was mandatory for beta-Gal translocation in vivo, transmembrane pores were not formed as concluded from the trypan blue exclusion method. These results altogether indicated that protein uptake both in vitro with LUV and in vivo with HeLa cells was mainly, if not solely, dependent on negative transmembrane potential across the bilayer, which suggests a physical mechanism governed by electrostatic interactions between pep-1 (positively charged) and membranes (negatively charged).