Histone posttranslational modifications play fundamental roles in the regulation of double-stranded DNA break (DSB) repair. RNF20/RNF40-mediated monoubiquitination of histone H2B on lysine 120 (H2Bub) has been suggested as a potential mediator of DSB repair, although the nature and function of this posttranslational modification remain enigmatic. In this report, we demonstrate that RNF20 and RNF40 are required for DSB repair leading to homologous recombination (HR) and class switch recombination, a process driven by nonhomologous end joining (NHEJ), in mouse B cells. These findings suggest a role for RNF20 and RNF40 in DSB repair proximal to NHEJ/HR pathway choice and likely in the signaling of DSBs. We found that DSBs led to a global increase in H2Bub but not the transcription-associated posttranslational modifications H3K4me3 and H3K79me2. We also found that H2AX phosphorylation was dispensable for H2Bub and that ATM and ATR jointly regulate ionizing radiation (IR)-induced H2Bub. Together, our results suggest that RNF20, RNF40, and H2Bub may represent a novel pathway for DSB sensing and repair.