Methylation of histone tails plays an important role in chromatin structure and function. Previously, we reported that ESET/SETDB1 is a histone methyltransferase (HMTase). Here, we show that SETDB1 tightly associates with the human homolog of mAM, a murine ATFa-associated factor. Although recombinant ESET can methylate lysine 9 of histone H3 (H3-K9), its activity is severely compromised when compared to that of the ESET/mAM complex. mAM stimulates ESET enzymatic activity by increasing the Vmax and decreasing the Km. Importantly, mAM facilitates the ESET-dependent conversion of dimethyl H3-K9 to the trimethyl state both in vitro and in vivo. Chromatin-based transcription and ChIP analyses demonstrate that mAM enhances ESET-mediated transcriptional repression in a SAM-dependent manner, and this repression correlates with H3-K9 trimethylation at the promoter. Thus, our studies establish that promoter H3-K9 trimethylation is the cause of transcriptional repression and that mAM/hAM facilitates conversion of H3-K9 dimethyl to trimethyl by ESET/SETDB1.