Type VI secretion systems (T6SS) are bacteriophage-derived macromolecular machines responsible for the release of at least two proteins in the milieu, which are thought to form an extracellular appendage. Although several T6SS have been shown to be involved in the virulence of animal and plant pathogens, clusters encoding these machines are found in the genomes of most species of gram-negative bacteria, including soil, marine, and environmental isolates. T6SS have been associated with several phenotypes, ranging from virulence to biofilm formation or stress sensing. Their various environmental niches and large diversity of functions are correlated with their broad variety of regulatory mechanisms. Using a bioinformatic approach, we identified several clusters, including those of Vibrio cholerae, Aeromonas hydrophila, Pectobacterium atrosepticum, Pseudomonas aeruginosa, Pseudomonas syringae pv. tomato, and a Marinomonas sp., which possess typical -24/-12 sequences, recognized by the alternate sigma factor sigma 54 (σ(54) or σ(N)). σ(54), which directs the RNA polymerase to these promoters, requires the action of a bacterial enhancer binding protein (bEBP), which binds to cis-acting upstream activating sequences. Putative bEBPs are encoded within the T6SS gene clusters possessing σ(54) boxes. Using in vitro binding experiments and in vivo reporter fusion assays, we showed that the expression of these clusters is dependent on both σ(54) and bEBPs.