The ATP-binding cassette transporter family C 2 (Abcc2) is a member of efflux transporters involved in the biliary excretion of organic anions from hepatocytes. Post-translational regulation of Abcc2 has been implicated, although the molecular mechanism is not fully understood. In the present study, we performed yeast two hybrid screening to identify novel protein(s) which particularly interacts with the linker region of Abcc2 located between the N-terminal nucleotide binding domain and the last membrane spanning domain. The screening resulted in the identification of a series of SUMO-related enzymes and their substrates. In yeast experiments, all of these interactions were abolished by substituting the putative SUMO consensus site in the linker region (IKKE) in Abcc2 to IRKE. In vitro SUMOylation experiments confirmed that the Abcc2 linker was a substrate of Ubc9-mediated SUMOylation. It was also found that the IKKE sequence is the target of SUMOylation, since a mutant with IKKE is substituted by IRKE was not SUMOylated. Furthermore, we demonstrated for the first time that Abcc2, endogenously expressed in rat hepatoma derived McARH7777 cells, is SUMOylated. Suppression of endogenous Ubc9 by siRNA resulted in a selective 30% reduction in Abcc2 protein expression in the post-nuclear supernatant, while subcellular localization of Abcc2 confirmed by semi-quantitative immunofluorescence analysis was minimally affected. This is the first demonstration showing the regulation of ABC transporter expression by SUMOylation.