Abstract The bridging integrator 1 (BIN1) protein was originally identified as a cellular adaptor protein that interacts with the transcription factor c-MYC and inhibits cell proliferation. However, BIN1 inhibits tumor cell growth by both a BIN1 MYC-binding domain (MBD)-dependent mechanism and an MBD-independent mechanism. Consistently, BIN1 promotes skeletal muscle differentiation in vitro where c-MYC is not detectable, suggesting that an undefined BIN1-interacting protein other than c-MYC determines BIN1-mediated growth arrest in an MYC-independent manner. Here we show the functional interplay between BIN1 and the RB1 tumor suppressor for growth arrest. BIN1 interacts with RB1 in differentiated (i.e. growth-arrested) C2C12 skeletal myotube cells, whereas such interaction was largely reduced in undifferentiated (i.e. actively proliferating) C2C12 myoblast cells. Acting through the coiled-coil BAR domain, an essential effector region for BIN1-mediated growth suppression, BIN1 physically interacts with the C pocket domain of RB1 in vitro and in vivo. The RB1-BIN1 protein-protein interaction is functionally germane. First, adenovirus E1A viral oncoprotein, which is known to stimulate cell proliferation in rodent fibroblast model systems by interacting with RB1, disrupted the RB1-BIN1 interaction in vitro. Second, the cancer suppression mediated by overexpressed BIN1 was partly attenuated by the co-transfection of short-hairpin RNA against RB1. Third, an MBD-independent BIN1-associated transcriptional corepressor activity was attenuated when endogenous RB1 was silenced. Fourth, serum stimulation-dependent phosphorylation of the RB-C pocket domain at serine 807/811, critical phosphorylation sites for RB1 inactivation by cyclin-dependent kinases 4 and 6 (CDK4/6), was largely reduced in BIN1-proficient cells. Conversely, the effect of PD-0332991, a chemical inhibitor of CDK4/6, on the growth suppression of RB-proficient cancer cells was more profound in BIN1-deficient cancer cells. Collectively, these data suggest that BIN1 and RB1 physically interact with one another and cooperate to induce growth arrest during cellular differentiation and cancer suppression. Citation Format: Watson P. Folk, Alpana Kumari, Erica Cassimere, Joanna Johnson, Daitoku Sakamuro. Physical and functional interactions between two tumor suppressors, BIN1 and RB1. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr B03.