RUNX3 is a tumor suppressor gene localized in 1p36. In various human tumors, the region is frequently inactivated through hypermethylation, histone modulation and other processes. Recent studies have suggested that loss of RUNX3 expression is involved in stomach, colon and breast cancer. However, the relationship between RUNX3 expression and giant cell tumor of the bone (GCTB) remains elusive. The aim of our study was to elucidate the roles of RUNX3 expression in carcinogenesis and progression of giant cell tumor of the bone. The levels of RUNX3 mRNA and protein were evaluated in human GCTB specimens and cell lines. To assess RUNX3 methylation we employed methylation-specific polymerase chain reaction using GCTB specimens and cell lines. In addition, to examine the roles of RUNX3 in giant cell tumor of the bone, GCTB cells were transfected with pcDNA3.1-RUNX3 (RUNX3 was cloned into the pcDNA3.1 plasmid). Flow cytometry (FCM) was used to analyze the apoptosis and cell cycle. The mobility of cells was tested by transwell migration assay. The expression rates of RUNX3 in patients with GCTB were significanly lower than normal bone tissues. Thirty of 47 human cancer specimens exhibited suppression (P<0.05). Down-regulation of RUNX3 mRNA in the same GCTB cell lines was associated with RUNX3 DNA methylation. In in vitro experiments, exogenous expression of RUNX3 strongly inhibited cell growth in GCTB by MTT (P<0.05), induced apoptosis as evidenced by Annexin V-FITC and increased G1 phase ratio by PI (P<0.05). Transwell migration assay showed that less RUNX3 positive cells migrated to the lower side of the membrane than negative ones (P<0.05). These results show that RUNX3 is a tumor suppressor in GCTB. RUNX3 DNA methylation may be the molecular basis for its lower expression. These data may be applied in GCTB for diagnostics and therapeutics.