The type III secretion system (T3SS) is a key pathogenicity factor of many Gram-negative plant and animal pathogens and is also present in some symbiotic bacteria. T3SSs are highly conserved, complex molecular injection machines that translocate bacterial effector proteins directly into eukaryotic cells. Recent genetic, biochemical and structural studies revealed that the activity of the T3SS is regulated by common mechanisms in different bacterial species. In many cases, type III secretion is activated upon host cell contact and depends on specific control proteins, including transcriptional regulators and type III secretion chaperones.