Segregation of homologous chromosomes in meiosis I is tightly regulated by their physical links, or crossovers (COs), generated from DNA double-strand breaks (DSBs) through meiotic homologous recombination. In budding yeast, three ZMM (Zip1/2/3/4, Mer3, Msh4/5) proteins, Zip2, Zip4, and Spo16, form a "ZZS" complex, functioning to promote meiotic recombination via a DSB repair pathway. Here, we identified the mammalian ortholog of Spo16, termed SPO16, which interacts with the mammalian ortholog of Zip2 (SHOC1/MZIP2), and whose functions are evolutionarily conserved to promote the formation of COs. SPO16 localizes to the recombination nodules, as SHOC1 and TEX11 do. SPO16 is required for stabilization of SHOC1 and proper localization of other ZMM proteins. The DSBs formed in SPO16-deleted meiocytes were repaired without COs formation, although synapsis is less affected. Therefore, formation of SPO16-SHOC1 complex-associated recombination intermediates is a key step facilitating meiotic recombination that produces COs from yeast to mammals.