BACKGROUND: Acquired immunodeficiency syndrome (AIDS) cannot be completely cured, and the main obstacle is the existence of viral reservoirs. However, we currently do not fully understand the molecular mechanisms by which HIV-1 latency is established and maintained. METHODS: Here, based on engineered chromatin immunoprecipitation (enChIP) technology that using FLAG-tagged zinc finger nucleic acid proteins (FLAG-ZFP) that bind to the HIV-1 L region and chromatin immunoprecipitation, we identified RYBP as a new HIV-1 latency-promoting gene. The effect of RYBP on HIV-1 latency was explored in multiple cell lines and primary latency models through gene knockout methods. Western blot and chromatin immunoprecipitation (ChIP) were used to explore the molecular mechanism of RYBP in promoting HIV-1 latency. RESULTS: Disruption of RYBP gene can activate latent HIV-1 in different latent cell lines and primary latent cell models. Mechanistically, the HIV-1 long terminal repeats (LTR) region binding protein Yin Yang 1 (YY1) can recruit RYBP to the HIV-1 L region. Then, RYBP can further recruit KDM2B, thereby promoting the increased ubiquitination level of H2AK119 and decreases the level of H3K4me3, to decrease HIV-1 L transcriptional elongation and enter a latent state. At the same time, during the stage of viral transcription and replication, Tat protein can inhibit the expression of RYBP, promoting viral transcription and replication. Finally, we found that the H2AK119ub inhibitor PRT4165 can promote latent HIV-1 activation and has good synergy with reported latent reactivating agents. CONCLUSION: These results provide mechanistically new insights into a critical role of RYBP in the regulation of histone modification and H2AK119ub may be directly targeted to control HIV reservoirs.