During the differentiation of the osteoclast, it transform from a mono-nucleated cell to multinucleated cell by fusion of its precursors. Although, cell fusion is a complicated process which likely involves many regulating proteins, only a few osteoclast fusion regulating proteins were identified. We have identified dual-specificity tyrosine-phosphorylation regulated kinase 2 (Dyrk2) as a novel regulator of the osteoclast fusion process. Dyrk2 belongs to a family of protein kinases whose members are involved in cell cycle and cytoskeleton organization. We found that Dyrk2 mRNA expression increases, 48 hours after induction of osteoclast differentiation in vitro, a time point were fusion commence. Dyrk2 knock-down, increases osteoclast nuclei numbers, but not osteoclast numbers and area, in differentiating RAW 264.7 cells and primary bone marrow derived monocytes (BMMs), While, overexpression of Dyrk2 decreases the number of osteoclast and osteoclast nuclei numbers suggesting it is involved in regulation of the fusion process per se. In order to further determine if Dyrk2 is involved in the fusion mechanism and not in earlier differentiation steps that lead to a fusion competent state, we investigated its role in giant macrophage fusion which are induced by different signaling pathways but share a similar fusion mechanism. Knockdown of Dyrk2 in giant macrophages increased the number of nuclei per macrophage, supporting a more general role of Dyrk2 in limiting the fusion process. While attempting to uncover the mechanism at which Dyrk2 regulates fusion we found that the rate of fusion of multinuclear cells is faster in Dyrk2 deficient osteoclasts. Moreover an ectopic expression of a GFP-tagged Dyrk2 showed co localization of Dyrk2 and actin in osteoclasts actin rings known to regulate osteoclast fusion. Taken together, our data reveal a novel role for Dyrk2 as a negative regulator of osteoclasts and giant macrophage fusion likely through the modulation of the osteoclast cytoskeleton.