Meiotic DNA double-strand breaks (DSBs) initiate homologous recombination and are crucial for ensuring proper chromosome segregation. In mice, ANKRD31 recently emerged as a regulator of DSB timing, number, and location, with a particularly important role in targeting DSBs to the pseudoautosomal regions (PARs) of sex chromosomes. ANKRD31 interacts with multiple proteins, including the conserved and essential DSB-promoting factor REC114, so it was hypothesized to be a modular scaffold that “anchors” other proteins together and to meiotic chromosomes. To determine if and why the REC114 interaction is important for ANKRD31 function, we generated mice withAnkrd31mutations that either reduced (missense mutation) or eliminated (C-terminal truncation) the ANKRD31–REC114 interaction without diminishing contacts with other known partners. A complete lack of the ANKRD31–REC114 interaction mimicked anAnkrd31null, with delayed DSB formation and recombination, defects in DSB repair, and altered DSB locations including failure to target DSBs to the PARs. In contrast, when the ANKRD31–REC114 interaction was substantially but not completely disrupted, spermatocytes again showed delayed DSB formation globally, but recombination and repair were hardly affected and DSB locations were similar to control mice. The missenseAnkrd31allele showed a dosage effect, wherein combining it with the null or C-terminal truncation allele resulted in intermediate phenotypes for DSB formation, recombination, and DSB locations. Our results show that ANKRD31 function is critically dependent on its interaction with REC114, and that defects in ANKRD31 activity correlate with the severity of the disruption of the interaction.