A well-characterized early histone modification that is triggered by DNA is the phosphorylation and ubiquitination of the histone variant H2AX. This occurs minutes after the formation of DNA double strand breaks and leads to enhanced access by proteins, including NBS1 and others, that participate in homologous recombination-mediated repair However, it is likely that another histone modification could be involved in this DNA repair pathway because H2AX knock out cells still display a normal localization of RAD51, a key protein in HRR, to sites of DSBs and exhibit significant HRR activity. We report here that, after exposure to ionizing radiation, H2B is mono-ubiquitinated independently of H2AX modification and plays a critical role in RAD51-mediated HRR of DSBs. RNF20, an E3 ubiquitin ligase implicated in chromatin remodeling for transcription, forms IR-induced nuclear foci and promotes H2B mono-ubiquitination 1.5 hours post-irradiation, and this is followed by the release of H2B and H3 from chromatin. Surprisingly, the formation of IR-induced foci containing RAD51 and BRCA1 was substantially suppressed by decreased RNF20 expression or by a mutation of the ubiquitinated amino acid residue in H2B. Consistent with these data, depletion of RNF20 interfered with the accumulation of RAD51 and BRCA1 in chromatin and resulted in a pronounced reduction in HRR. As a result, RNF20 down-regulation significantly enhanced sensitivity to mitomycin C and IR. Moreover, RNF20-deficient phenotypes were corrected in part by chromatin decondensation using a histone deacetylase inhibitor, but IR-induced H2B ubiquitination was not affected by the addition of transcription inhibitors, suggesting the existence of transcription-independent chromatin reorganization activity at DSB sites by RNF20.