Mutations or deletions of the TRPS1 gene on human chromosome 8q.24.1 cause the tricho-rhino-phalangeal syndromes (TRPS), which are characterized by craniofacial and skeletal malformations. The gene encodes a transcription factor that functions as a repressor for GATA-mediated transcription. The activity of transcription factors is often controlled by posttranslational modifications. We show here that TRPS1 is SUMOylated at multiple sites, both in vivo and in vitro, through interaction with UBC9. Overexpression of wild-type UBC9 enhances TRPS1-mediated transcriptional repression. In contrast, a SUMOylation-deficient UBC9 mutant, which nevertheless still binds TRPS1, has no effect. Of the five potential TRPS1 SUMO-target sites, which were predicted based on a minimal SUMOylation consensus sequence (MCS), two are located within the C-terminal repression domain (RD) at lysine residues 1192 (termed S4) and 1201 (S5). S5 was identified as the major acceptor site within this region, and a point mutation of S5 strongly decreases TRPS1-RD-mediated transcriptional repression. Additional mutation of S4 results in abrogation of SUMOylation at the TRPS1-RD and almost complete loss of the repressive properties of TRPS1. These results identify SUMOylation at the TRPS1-RD as a major mechanism that regulates the function of TRPS1.