AbstractRor2 is an orphan receptor tyrosine kinase that plays crucial roles in developmental morphogenesis, particularly of the skeleton. We have identified human Ror2 as a novel regulator of canonical Wnt signaling in osteoblastic (bone-forming) cells with selective activities, enhancing Wnt1 but antagonizing Wnt3. Immunoprecipitation studies demonstrated physical interactions between human Ror2 and mammalian Wnt1 and Wnt3. Functionally, Ror2 antagonized Wnt1- and Wnt3-mediated stabilization of cytosolic β-catenin in osteoblastic cells. However, Ror2 had opposing effects on a more distal step of canonical Wnt signaling: it potentiated Wnt1 activity but inhibited Wnt3 function as assessed by changes in Wnt-responsive reporter gene activity. Despite binding to Ror2, neither Wnt1 nor Wnt3 altered receptor activity as assessed by levels of Ror2 autophosphorylation. The ability of Ror2 to regulate canonical Wnt signaling in osteoblastic cells should have physiological consequences in bone, because Wnt signaling is known to modulate osteoblast survival and differentiation. Expression of Ror2 mRNA was highly regulated in a biphasic manner during human osteoblast differentiation, being virtually undetectable in pluripotent stem cells, increasing 300-fold in committed preosteoblasts, and disappearing again in osteocytes. Furthermore, Ror2 expression in osteoblasts was suppressed by the Wnt antagonist, secreted frizzled-related protein 1. The regulated expression of Ror2 during osteoblast differentiation, its inverse expression pattern with secreted frizzled-related protein 1, and its ability to modulate Wnt signaling in osteoblastic cells suggest that Ror2 may regulate bone formation.