A wide variety of bacterial pathogens secrete a unique class of proteins that attack target cell membranes and form transmembrane oligomeric pores with distinct β-barrel structural scaffolds. Owing to their specific mode of action and characteristic structural assembly, these proteins are termed as β-barrel pore-forming toxins (β-PFTs). The most obvious consequence of such pore-forming activity of bacterial β-PFTs is the permeabilization of cell membranes, which eventually leads to cell death. Bacterial β-PFTs have been studied extensively for nearly past four decades, and their mechanisms of actions have revealed some of the most enigmatic aspects of the protein structure-function paradigm. In most of the cases, β-PFTs are released by the bacteria as water-soluble monomeric precursors, which upon encountering target cell membranes assemble into membrane-inserted oligomeric pores. Structural descriptions are now documented for the water-soluble precursor forms, as well as for the membrane-anchored oligomeric pores of many β-PFTs. These studies have revealed that β-PFTs undergo a series of well-orchestrated structural rearrangements during membrane pore formation. Nevertheless, mechanisms that trigger and regulate distinct steps of the pore-formation processes still remain obscure. Here, we discuss our current understanding regarding structure-function mechanisms in the β-PFT family, with particular emphasis on some of the unsolved issues associated with the β-barrel pore-formation mechanism.