Aims: To investigate the effects of the organic solvent dimethyl sulfoxide (DMSO) on the expression of a citrate-inducible gene, encoding a putative tricarboxylate transporter, in Agrobacterium tumefaciens. Methods and Results: By two-dimensional gel electrophoresis, we discovered a putative tricarboxylate transporter named ActC, whose expression was downregulated by DMSO. The expression of actC is also induced by tricarboxylates but not affected by other organic acids of the TCA cycle. Intriguingly, transcriptional activation of actC by citrate is compromised in the presence of DMSO. Furthermore, expression of actC is abolished by deletion of actDE, encoding a putative two-component regulatory system upstream of the actCBA gene cluster. Conclusions: actC is a citrate-inducible gene that is repressed by DMSO and whose expression is likely regulated by a two-component system. Significance and Impact of the Study: This study provides useful information as to a potential DMSO-regulatory system of A. tumefaciens or other soil bacteria when encountering DMSO in nature. In addition, DMSO-regulated genes should be taken into account for studies in which bacterial cultures were treated with compounds dissolved in DMSO.