Ascorbic acid (AA) plays a key role in the regulation of differentiation and activation of osteoclast (OCL). It was reported that AA might induce the formation of OCL in co-cultures of mouse bone marrow cells and ST2 cells, but it is not clear whether AA has a direct impact on the OCL precursors. The purpose of this study was to examine the effect of AA on the differentiation of OCL precursor RAW264.7 cells, cultured with receptor-activated nuclear factor kappaB ligand (RANKL). The results showed that AA remarkably inhibited the cell proliferation at a higher concentration and RANKL alone is sufficient for osteoclastogenesis. The expression of carbonic anhydrase (CAII) mRNA and protein, the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs), and the percentage area of resorption lacunae induced by RANKL were decreased when AA was added to the cultures. The results demonstrate that AA inhibits RANKL-induced differentiation of OCL precursor cells into mature OCL and reduces the formation of bone resorption pits in vitro.