The efficient and timely resolution of DNA recombination intermediates is essential for bipolar chromosome segregation. Here, we show that the specialized chromosome segregation patterns of meiosis and mitosis, which require the coordination of recombination with cell-cycle progression, are achieved by regulating the timing of activation of two crossover-promoting endonucleases. In yeast meiosis, Mus81-Mms4 and Yen1 are controlled by phosphorylation events that lead to their sequential activation. Mus81-Mms4 is hyperactivated by Cdc5-mediated phosphorylation in meiosis I, generating the crossovers necessary for chromosome segregation. Yen1 is also tightly regulated and is activated in meiosis II to resolve persistent Holliday junctions. In yeast and human mitotic cells, a similar regulatory network restrains these nuclease activities until mitosis, biasing the outcome of recombination toward noncrossover products while also ensuring the elimination of any persistent joint molecules. Mitotic regulation thereby facilitates chromosome segregation while limiting the potential for loss of heterozygosity and sister-chromatid exchanges. Display omitted ► Mus81-Mms4 and Yen1 endonucleases collaboratively resolve DNA joint molecules in meiosis ► Cdc5 activates Mus81-Mms4 during meiosis I through the phosphorylation of Mms4 ► Phosphorylation inhibits Yen1 until the onset of meiosis II ► Yen1/GEN1 and Mus81-Mms4/EME1 are specifically activated during M phase of mitosis Endonucleases resolve DNA recombination intermediates at different stages to promote and prevent crossing over in meiosis and mitosis, respectively.