Osteoclasts are the key target cells for cadmium (Cd)-induced bone metabolism diseases, while Rho GTPases play an important role in osteoclast differentiation and bone resorption. To identify new therapeutic targets of Cd-induced bone diseases; we evaluated signal transduction through Rho GTPases during osteoclast differentiation under the influence of Cd. In osteoclastic precursor cells, 10 nM Cd induced pseudopodia stretching, promoted cell migration, upregulated the levels of Cdc42, and RhoQ mRNAs and downstream Rho-associated coiled-coil kinase 1 (ROCK1) and ROCK2 proteins, and downregulated the actin-related protein 2/3 (ARP2/3) levels.Cd at 2 and 5 μM shortened the pseudopodia, inhibited cell migration, and decreased ROCK1, ROCK2, and ARP2/3 protein levels; Cd at 5 μM also reduced the mRNA expression levels of Rac1, Rac2, and RhoU mRNAs and decreased the level of phosphorylated (p)-cofilin. In osteoclasts, 10 nM Cd induced the formation of sealing zones, slightly upregulated Cdc42 mRNA levels and ROCK2 and ARP2/3 protein levels and significantly reduced p-cofilin levels. Cd at 2 μM and 5 μM Cd blocked the fusion of precursor cells; and 5 μM Cd downregulated the expression levels of RhoB, Rac1, Rac3, and RhoU mRNAs, and ROCK1, p-cofilin and ARP2/3 protein levels, significantly. In vivo, Cd (at 5 or 25 mg/L) increased the levels of key proteins RhoA, Rac1/2/3, Cdc42, and RhoU and their mRNAs in bone marrow cells. In summary, the results suggested that Cd affected the differentiation process of osteoclast and altered the expression of several Rho GTPases, which might be crucial targets of Cd during the differentiation of osteoclasts.