The microphthalmia transcription factor MITF plays important roles in several cell lineages including retinal and neural crest-derived pigment cells. Previous reports have shown that besides its regulation at the transcriptional level, MITF is also regulated post-translationally by phosphorylation and ubiquitination which affect the protein's activity and stability. Here we demonstrate that in addition, MITF is modified in melanoma cells by small ubiquitin-like modifier (SUMO). In vitro assays further show that sumoylation occurs at two lysine residues, K182 and K316, and depends on SUMO E1 activating enzyme (SAE I/SAE II) and E2 conjugating enzyme (Ubc9). Interestingly, MITF with double lysine 182/316 to arginine mutations, although displaying normal DNA binding, stability and nuclear localization, shows a substantial increase in the transcriptional stimulation of promoters containing multiple but not single MITF binding sites. MITF containing the double lysine-to-arginine substitution also shows enhanced cooperation with Sox10 on the Dct promoter. We conclude that SUMO modification of MITF regulates the protein's transcriptional activity especially with respect to synergistic activation. The results suggest that sumoylation plays a significant role among the multiple mechanisms that regulate MITF during development and in adulthood.