Pili of gram-negative pathogens play an important role in pathogenesis, and the genetics and mechanism of pilus assembly in these organisms is now well understood . In contract, the pilus assembly pathways and funtions in Gram-positive bacteria remain to be characterized. Gram-positive microbes use the cell wall peptidoglycan as a surface organelle for the covalent attachment of proteins; a strategy that involves sorting signals of surface protein precursors and sortase, a transpeptidase that cleaves sorting signals and links the C-terminus of surface proteins via an amide bond to the peptidoglycan cross-bridge. Using Corynebacterium diphtheriae as model system, we showed that pili of C. diphtheriae are composed of three pilin subunits, SpaA, SpaB and SpaC. SpaA, the major pilin protein, is distributed uniformly along the pilus shaft, while SpaB is observed at regular intervals and SpaC seems positioned at the pilus tip. Pilus assembly is proposed to occur by a mechanism of ordered cross-linking, whereby pilin-specific sortase enzymes cleave precursor proteins at sorting signals and involve the side chain amino groups of pilin motif sequences to generate links between pilin subunits. This covalent tethering of adjacent pilin subunits appears to have evolved in many gram-positive pathogens that encode sortase and pilin subunit genes with sorting signals and pilin motifs. Using an ex-vivo culture model, we demonstrated that corynebacterial pili are involved in the bacterial adherence to host cell tissues, implicating the role of pili in pathogenesis.