During meiosis, at least one crossover must occur in each chromosome pair to ensure their accurate segregation. However, the mechanism of crossover formation is poorly understood. Here, we report a previously uncharacterized recombination protein, C12ORF40/REDIC1, essential for meiotic crossover formation. A homozygous frameshift mutation in C12orf40 was identified in two genetically unrelated infertile men with meiotic arrest. Subsequent analysis showed that REDIC1 forms discrete foci in the paired regions of homologous chromosomes depending on strand invasion. Mouse models mimicking the patients’ mutation showed reductions in crossover and bivalent formation, leading to spermatogenic failure. Notably, mutant spermatocytes displayed reduced numbers of MSH4 and TEX11 foci, accompanied by synaptic defects. Correspondingly, these phenotypes were also observed in our patients. Finally, biochemical results showed that the identified mutation impairs its ability to bind branched recombination intermediates. Therefore, our findings reveal a crucial role for C12ORF40/REDIC1 in meiotic crossover formation by stabilizing the recombination intermediates, providing prospective molecular targets for the clinical diagnosis and therapy of infertility.