Peg3 (paternally expressed gene 3) encodes a DNA-binding protein that functions as a transcriptional repressor. Recent studies revealed that PEG3 binds to Msl1 (male-specific lethal 1) and Msl3, the two main components of the MSL complex. In the current study, we investigated potential roles of Peg3 in controlling its downstream genes through H4K16ac, the histone modification by the MSL complex. According to the results, complete removal of PEG3 resulted in up-regulation of Msl1 and Msl3, and subsequently an increase in the global levels of H4K16ac, confirming PEG3 as a transcriptional repressor for MSL during mammalian development. Genome-wide analyses further revealed that about 10% of the entire gene catalogue was affected in the MEF cells lacking PEG3, displaying the increased levels of H4K16ac in their promoter regions. The expression levels of a small subset of the affected genes were up-regulated in the MEF cells lacking PEG3. Interestingly, three Hox clusters also exhibited changes in the levels of H4K16ac, suggesting potential roles of PEG3 and MSL in the regulation of Hox clusters. Overall, the current study reports that Peg3 may control its downstream genes through mammalian MSL.