Biomolecules such as peptides, proteins, and nucleic acids generally cannot cross a cell membrane by passive diffusion. Nevertheless, cell-penetrating peptides (CPPs), bacterial protein toxins, certain eukaryotic proteins, viruses, and many synthetic drug delivery vehicles have been shown to enter the cytosol of eukaryotic cells with varying efficiencies. They generally enter the cell by one or more of the endocytic mechanisms and are initially localized inside the endosomes. But how they cross the endosomal membrane to reach the cytosol (i.e., endosomal escape) has been a mystery for decades, and this knowledge gap has been a major bottleneck for the development of efficient drug delivery systems. In addition, many bacterial and eukaryotic proteins are transported across the plasma membrane in their native states into the periplasmic/extracellular space through the twin-arginine translocation (TAT) and unconventional protein secretion (UPS) systems, respectively. Again, the mechanisms underpinning these protein export systems remain unclear.In this Account, I introduce a previously unrecognized, fundamental membrane translocation mechanism which we have termed the vesicle budding-and-collapse (VBC) mechanism. Through VBC, biomolecules of diverse sizes and physicochemical properties autonomously translocate across cell membranes topologically (i.e., from one side to the other side of the membrane) but not physically (i.e., without going through the membrane). We have demonstrated that CPPs and bacterial protein toxins escape the endosome by the VBC mechanism in giant unilamellar vesicles as well as live mammalian cells. This advance resulted from studies in which we labeled the biomolecules with a pH-sensitive, red-colored dye (pHAb) and phosphatidylserine with a pH-insensitive green dye (TopFluor) and monitored the intracellular trafficking of the biomolecules in real time by confocal microscopy. In addition, by enlarging the endosomes with a kinase inhibitor, we were able to visualize the structural changes of the endosomes (i.e., endosomal escape intermediates) as they went through the VBC process. I postulate that bacterial/viral/eukaryotic proteins, nonenveloped viruses, and synthetic drug delivery vehicles (e.g., polyplexes, lipoplexes, and lipid nanoparticles) may also escape the endosome by inducing VBC. Furthermore, I propose that VBC may be the mechanism that drives the bacterial TAT and eukaryotic UPS systems. Our findings fill a long-standing gap in cell biology and provide guiding principles for designing more efficient drug delivery vehicles.