Wnt signaling is intrinsically involved in diverse cellular activities during cell differentiation, early embryonic development and organogenesis. Although much is known regarding the effects of Wnt signaling in the developing skeletal system, its role during regeneration remains unclear. Herein, we show transcriptional activation of specific members and target genes of the Wnt signaling pathway. Specifically, all of the Wnt signaling members and target genes analyzed were found to be upregulated during the early stages of fracture repair, with the exception of LEF1 whose expression was downregulated. In addition, spatial expression analysis of Dishevelled (Dvl) and beta-catenin in the fracture callus revealed an identical pattern of expression with both proteins localizing in osteoprogenitor cells of the periosteum, osteoblasts and proliferating/pre-hypertrophic chondrocytes. Further, in vitro knockdown of all three Dvl isoforms in chondrocytes using small interfering RNAs (siRNA) leads to partial inhibition of cell proliferation and differentiation, decreased expression of chondrogenic markers (ColII, ColX, Sox9) and suppressed nuclear accumulation of unphosphorylated beta-catenin. Taken together, these data verify our previous finding that the Wnt signaling pathway is activated during bone regeneration, by characterizing the temporal and spatial expression of a broad spectrum of Wnt-signaling molecules. Our data also suggest that all three Dvl isoforms, acting through the Wnt canonical pathway, are critical regulatory molecules for chondrocyte proliferation and differentiation.