Perfringolysin O (PFO) is a transmembrane (TM) β-barrel protein that inserts into mammalian cell membranes. Once inserted into membranes， PFO assembles into pore-forming oligomers containing 30-50 PFO monomers. These form a pore of up to 300 Å， far exceeding the size of most other proteinaceous pores. In this study， we found that altering PFO TM segment length can alter the size of PFO pores. A PFO mutant with lengthened TM segments oligomerized to a similar extent as wild-type PFO， and exhibited pore-forming activity and a pore size very similar to wild-type PFO as measured by electron microscopy and a leakage assay. In contrast， PFO with shortened TM segments exhibited a large reduction in pore-forming activity and pore size. This suggests that the interaction between TM segments can greatly affect the size of pores formed by TM β-barrel proteins. PFO may be a promising candidate for engineering pore size for various applications.