Abstract: Objective To identify a novel retinoblastoma protein(RB)-associated protein(RBAP 96) and to explore the impact of RBAP96 on radiosensitivity of human breast cancer cells. Methods An in vivo and in vitro association of RBAP96 with RB was determined by immunoprecipitation-Western blotting and GST pull-down assay. Protein expression was measured by Western blot assay. Cellular survival was evaluated by using a colony formation assay. Results In both in vitro and in vivo assays, we found that the RBAP96 and RB interaction required a ⁵¹³LXCXE⁵¹⁷ motif on the RBAP96 protein and an intact A/B binding pocket of RB. RBAP96 enhances RB-mediated transcriptional repression. Finally, enforced expression of RBAP96 caused an elevated radiosensitivity of human breast cancer cells bearing wtRB, but did not affect radiosensitivity of breast cancer cells bearing mutant RB. Expression of a full-length RBAP96 with an ⁵¹³LXCXE⁵¹⁷ inactivating mutation(LXCXE?RXRXH) failed to result in any radiosensitivity alteration. Conclusion This study for the first time characterizes a novel RB-interacting protein RBAP96 and demonstrates that enforced expression of RBAP96 causes an increase of RBAP96-mediated transcription activation and radiosensitivity via a RB-interacting dependent manner.