Genetic studies in Caenorhabditis elegans identified lin-9 to function together with the retinoblastoma homologue lin-35 in vulva differentiation. We have now identified a human homologue of Lin-9 (hLin-9) and provide evidence about its function in the mammalian pRB pathway. hLin-9 binds to pRB and cooperates with pRB in flat cell formation in Saos-2 cells. In addition, hLin-9 synergized with pRB and Cbfal to transactivate an osteoblast-specific reporter gene. In contrast, hLin-9 was not involved in pRB-mediated inhibition of cell cycle progression or repression of E2F-dependent transactivation. Consistent with these data, hLin-9 was able to associate with partially penetrant pRB mutants that do not bind to E2F, but retain the ability to activate transcription and to promote differentiation. hLin-9 can also inhibit oncogenic transformation, dependent on the presence of a functional pRB protein. RNAi-mediated knockdown of Lin-9 can substitute for the loss of pRB in transformation of human primary fibroblasts. These data suggest that hLin-9 has tumor-suppressing activities and that the ability of hLin-9 to inhibit transformation is mediated through its association with pRB.