Proper and timely repair of DNA double-strand breaks (DSBs) is critical for preservation of genome integrity. While DSBs are repaired through a balance of canonical nonhomologous end-joining (NHEJ) and homologous recombination (HR) pathways, this balance is frequently disrupted in HR-deficient cancer cells, causing misuse of NHEJ that destabilizes the genome. Understanding the balance between HR and NHEJ, and the factors that could modulate this balance, informs our understanding of genome integrity, tumorigenesis, and response to clinical therapy. The HR-NHEJ balance is regulated by several factors, including cell cycle-dependent phosphorylation events and chromatin remodeling. Our group and others previously published that the histone acetyltransferase Tat-Interacting Protein 60kDA (TIP60) limits the accumulation of p53-Binding Protein 1 (53BP1) at DSBs to promote the loading of Breast Cancer Type 1 Susceptibility Protein (BRCA1) and repair by HR, but the mechanism was unclear. Here we report that Tip60 conditional knockout causes meiotic HR defects in a BRCA1-independent manner, that its pro-HR activity is regulated by cell cycle-dependent phosphorylation, and that its acetylation of histones H4 and H2A.Z suppresses 53BP1 occupancy at DSBs. This body of work sheds light on the specific mechanisms by which this multi-faceted acetyltransferase directs DSB repair pathway choice.