Type III secretion systems (T3SSs) are complex bacterial structures that provide gram-negative pathogens with a unique virulence mechanism enabling them to inject bacterial effector proteins directly into the host cell cytoplasm, bypassing the extracellular milieu. Although the effector proteins vary among different T3SS pathogens, common pathogenic mechanisms emerge, including interference with the host cell cytoskeleton to promote attachment and invasion, interference with cellular trafficking processes, cytotoxicity and barrier dysfunction, and immune system subversion. The activity of the T3SSs correlates closely with infection progression and outcome, both in animal models and in human infection. Therefore, to facilitate patient care and improve outcomes, it is important to understand the T3SS-mediated virulence processes and to target T3SSs in therapeutic and prophylactic development efforts.