Yin Yang 1 (YY1) is a well-known transcription factor that controls the expression of many genes and plays an important role in the occurrence and development of various cancers. We previously found that the human males absent on the first (MOF)-containing histone acetyltransferase (HAT) complex may be involved in regulating YY1 transcriptional activity; however, the precise interaction between MOF-HAT and YY1, as well as whether the acetylation activity of MOF impacts the function of YY1, has not been reported. Here, we present evidence that the MOF-containing male-specific lethal (MSL) HAT complex regulates YY1 stability and transcriptional activity in an acetylation-dependent manner. First, the MOF/MSL HAT complex was bound to and acetylated YY1, and this acetylation further promoted the ubiquitin–proteasome degradation pathway of YY1. The MOF-mediated degradation of YY1 was mainly related to the 146–270 amino acid residues of YY1. Further research clarified that acetylation-mediated ubiquitin degradation of YY1 mainly occurred through lysine 183. A mutation at the YY1K183 site was sufficient to alter the expression level of p53-mediated downstream target genes, such asCDKN1A(encoding p21), and it also suppressed the transactivation of YY1 onCDC6. Furthermore, a YY1K183R mutant and MOF remarkably antagonized the clone-forming ability of HCT116 and SW480 cells facilitated by YY1, suggesting that the acetylation–ubiquitin mode of YY1 plays an important role in tumor cell proliferation. These data may provide new strategies for the development of therapeutic drugs for tumors with high expression of YY1.