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The type VI secretion system (T6SS) is a widespread, versatile protein secretion system in pathogenic Proteobacteria. Several T6SSs are tightly regulated by various regulatory systems at multiple levels. However, the signals and/or regulatory mechanisms of many T6SSs remain unexplored. Here, we report on an acid-induced regulatory mechanism activating T6SS in Agrobacterium tumefaciens, a plant pathogenic bacterium causing crown gall disease in a wide range of plants. We monitored the secretion of the T6SS hallmark protein hemolysin-coregulated protein (Hcp) from A. tumefaciens and found that acidity is a T6SS-inducible signal. Expression analysis of the T6SS gene cluster comprising the imp and hcp operons revealed that imp expression and Hcp secretion are barely detected in A. tumefaciens grown in neutral minimal medium but are highly induced with acidic medium. Loss- and gain-of-function analysis revealed that the A. tumefaciens T6SS is positively regulated by a chvG/chvI two-component system and negatively regulated by exoR. Further epistasis analysis revealed that exoR functions upstream of the chvG sensor kinase in regulating T6SS. ChvG protein levels are greatly increased in the exoR deletion mutant and the periplasmic form of overexpressed ExoR is rapidly degraded under acidic conditions. Importantly, ExoR represses ChvG by direct physical interaction, but disruption of the physical interaction allows ChvG to activate T6SS. The phospho-mimic but not wild-type ChvI response regulator can bind to the T6SS promoter region in vitro and activate T6SS with growth in neutral minimal medium. We present the first evidence of T6SS activation by an ExoR-ChvG/ChvI cascade and propose that acidity triggers ExoR degradation, thereby derepressing ChvG/ChvI to activate T6SS in A. tumefaciens., Author Summary The bacterial type VI secretion system (T6SS) has diverse functions that contribute to the survival or fitness of many pathogenic bacteria in response to environmental cues. Numerous studies have shown that T6SS is highly regulated via multiple mechanisms, but the regulatory mechanisms of most T6SSs remain unknown. In this study, we discovered that T6SS is activated by acidity via an ExoR-ChvG/ChvI cascade in a plant pathogenic bacterium, Agrobacterium tumefaciens. Our data suggested that ExoR represses ChvG sensor kinase by physical interaction and the acid-induced degradation of periplasmic ExoR may derepress ChvG to activate T6SS by phosphorylation of the ChvI response regulator. The activation of T6SS by an acidic signal present in the wound site and intercellular space of plants implicates a role of T6SS during Agrobacterium–plant interactions. In view of the conservation of ExoR and ChvG/ChvI and wide distribution of T6SS in α-Proteobacteria, including many animal and plant pathogens and symbionts, the regulation of T6SS by the ExoR-ChvG/ChvI cascade may be a universal regulatory mechanism in these bacteria.