This study is to investigate the inhibitory effect of different concentrations of zoledronic acid on the activity of osteoclasts, to obtain characteristics on inhibitory effect and to find the lowest effective concentration of zoledronic acid. Marrow cells of C57 mice (6 weeks) were cultured in vitro. Osteoclasts were induced by single nuclear cells. According to the concentration of zoledronic acid, we set up the experimental group with five different concentrations, i.e. 1×10–8 mol/L, 1×10–7 mol/L, 1×10–6 mol/L, 1×10–5 mol/L, and 1×10–4 mol/L. The control group did not contain any bisphosphonate. By tartrate resistant acid phosphatase staining, the number of multinuclear cells, cells through the filter and bone resorption lacune were counted. Five days after the cultivation, the number of multinuclear cells in the experimental group decreased with the increase of concentration of zoledronic acid. Inhibition on the formation of osteoclasts in vitro was effective at 1×10–6 mol/L. At the concentration of 1×10–5 mol/L, the effect of inhibition on migration of osteoclast and bone resorption was more obvious. The effect was further enhanced at concentration of 1×10–4 mol/L. However, the concentration and inhibition curves were gradually mild. The inhibitory effect on different concentrations of zoledronic acid on the activity of osteoclasts was different. The inhibition effect was obvious at 1×10–6 mol/L. We should pay attention to administrate appropriate concentration of zoledronic acid in the clinical applications.