The C-terminal cytoplasmic tails of claudins are likely sites for interaction with proteins that regulate their function. We performed a yeast two-hybrid screen with the tail of human claudin-2 against a human kidney cDNA library and identified interactions with the PDZ3 domain of ZO-2 as well as ubiquitin-conjugating enzyme E2I (SUMO ligase-1) and E3 SUMO-protein ligase PIAS; the first is a predicted interaction, while the latter two are novel and suggest that claudin-2 is a substrate for SUMOylation. Using an in vitro SUMOylation assay, we identified K218 as a conjugation site on claudin-2; mutation of that lysine to arginine blocked SUMOylation. Stable expression of inducible GFP-SUMO-1 in MDCK cells resulted in decreased levels of claudin-2 protein by immunoblot and decreased claudin-2 membrane expression by immunofluorescence microscopy. We conclude that the cellular levels of claudin-2 may be modulated by SUMOylation, warranting further investigation of cellular pathways that regulate this modification in vivo.