The Wnt signaling pathway is an important regulator of cellular differentiation in a variety of cell types including osteoblasts. In this study, we investigated the impact of Wnt signaling on the function of human osteoblasts in relation to the stage of differentiation. Differentiating osteoblasts were created upon glucocorticoid (GC) treatment, whereas nondifferentiating osteoblasts were created by excluding GCs from the culture medium. GC-induced differentiation suppressed endogenous β-catenin levels and transcriptional activity. During GC-induced osteoblast differentiation, activation of Wnt signaling slightly decreased alkaline phosphatase activity, but strongly suppressed matrix mineralization. In addition, mRNA expression of several Wnt signaling related genes was strongly regulated during GC-induced osteoblast differentiation, including frizzled homolog 8, dickkopf homolog 1, and secreted frizzled-related protein 1. In contrast, in the absence of GC-induced differentiation, Wnt signaling acted positively by stimulating basal alkaline phosphatase activity. Interestingly, pre-stimulation of Wnt signaling in early osteoblasts enhanced their differentiation capacity later on during the GC-induced differentiation process. In conclusion, we showed a differentiation-dependent effect of Wnt signaling on osteoblasts. Wnt signaling stimulated early osteoblasts in their capacity to differentiate, whereas mature osteoblasts were strongly inhibited in their capacity to induce mineralization. Moreover, osteoblast differentiation suppressed endogenous Wnt signaling and changed the expression of multiple Wnt signaling related genes. J. Cell. Biochem. 104: 568–579, 2008. © 2008 Wiley-Liss, Inc.