Small ubiquitin-related modifiers, SUMO-2/3 and SUMO-1, are involved in gene regulation and nuclear structures. However, little is known about the roles of SUMO, in heterochromatin formation of mammalian cells. Here we demonstrate that SUMOs directly interact with human MCAF1, which forms complexes with either the methyl-CpG-binding protein MBD1 or SETDB1, which trimethylates histone H3 at lysine 9 (H3-K9) in the presence of MCAF1. Modification of MBD1 with either SUMO-2/3 or SUMO-1 facilitated the interaction between MBD1 and MCAF1, suggesting that SUMOylation links the methylation of DNA and histones. In a cultured human cell line, SUMOs were localized in MBD1- and MCAF1-containing heterochromatin regions that were enriched in trimethyl-H3-K9 and the heterochromatin proteins HP1beta and HP1gamma. Specific knockdown of either SUMO-2/3 or SUMO-1 induced dissociation of MCAF1, trimethyl-H3-K9, and the HP1 proteins from the MBD1-containing heterochromatin foci, suggesting a requirement for SUMOs for heterochromatin assembly. These findings provide insights into the roles of SUMOylation in the regulation of heterochromatin formation and gene silencing.