RASSF2 belongs to the Ras-association domain family (RASSF) of proteins, which may be involved in the Hippo signalling pathway. However, the role of RASSF2 in vivo is unknown. Here, we show that Rassf2 knockout mice manifest a multisystemic phenotype including haematopoietic anomalies and defects in bone remodelling. Bone marrow (BM) transplantation showed that Rassf2(-/-) BM cells had a normal haematopoietic reconstitution activity, indicating no intrinsic haematopoietic defects. Notably, in vitro differentiation studies revealed that ablation of Rassf2 suppressed osteoblastogenesis but promoted osteoclastogenesis. Co-culture experiments showed that an intrinsic defect in osteoblast differentiation from Rassf2(-/-) osteoblast precursors likely leads to both haematopoiesis and osteoclast defects in Rassf2(-/-) mice. Moreover, Rassf2 deficiency resulted in hyperactivation of nuclear factor (NF)-κB during both osteoclast and osteoblast differentiation. RASSF2 associated with IκB kinase (IKK) α and β forms, and suppressed IKK activity. Introduction of either RASSF2 or a dominant-negative form of IKK into Rassf2(-/-) osteoclast or osteoblast precursors inhibited NF-κB hyperactivation and normalized osteoclast and osteoblast differentiation. These observations indicate that RASSF2 regulates osteoblast and osteoclast differentiation by inhibiting NF-κB signalling.