The type II secretion system (T2SS) is used by Escherichia coli and other gram-negative bacteria to translocate many proteins, including toxins and proteases, across the outer membrane of the cell and into the extracellular space. Depending on the bacterial species, between 12 and 15 genes have been identified that make up a T2SS operon. T2SSs are widespread among gram-negative bacteria, and most E. coli appear to possess one or two complete T2SS operons. Once expressed, the multiple protein components that form the T2S system are localized in both the inner and outer membranes, where they assemble into an apparatus that spans the cell envelope. This apparatus supports the secretion of numerous virulence factors; and therefore secretion via this pathway is regarded in many organisms as a major virulence mechanism. Here, we review several of the known E. coli T2S substrates that have proven to be critical for the survival and pathogenicity of these bacteria. Recent structural and biochemical information is also reviewed that has improved our current understanding of how the T2S apparatus functions; also reviewed is the role that individual proteins play in this complex system.