Gram-negative bacteria have evolved diverse secretion systems/machineries to translocate substrates across the cell envelope. These various machineries fulfil a wide variety of functions but are also essential for pathogenic bacteria to infect human or plant cells. Secretion systems, of which there are seven, utilize one of two secretion mechanisms: (i) the one-step mechanism, whereby substrates are translocated directly from the bacterial-cytoplasm to the extracellular medium or into the eukaryotic-target cell; (ii) the two-step mechanism, whereby substrates are first translocated across the bacterial-inner membrane; once in the periplasm, substrates are targeted to one of the secretion systems that mediate the transport across the outer membrane and the release outside the bacterial cell. This review describes in details the main structural features of these secretion systems. Structural biology offers the possibility to understand the molecular mechanisms at play in the various secretion systems. It also helps to design specifically drugs that can block these machineries and thus attenuate the virulence of pathogenic bacteria.