Objective To investigate the effects of Betulinic acid（BA）on proliferation，migration， adherence properties，tumorsphere formation，stem cell property and osteoclast differentiation in human prostate can⁃ cer PC⁃3 cells in acidic microenvironment. Methods Culturing PC⁃3 cells in pH 7.4 and pH 6.5 medium. Effects of BA on proliferation and migration of PC⁃3 cells were assessed with the CCK⁃8 assay and Transwell assays，respectively. Effects of BA on stem cell characteristics of PC⁃3 cells were evaluated by cell⁃matrix adhesion assay. The osteoclasto⁃ genesis activity of BA in RAW264.7 cells was examined by osteoclast differentiation assay. The express levels of CD31 and CD44 in PC⁃3 cells were measured by qPCR and Western blot. Results The result of CCK⁃8 test showed that acidic medium significantly enhanced cell viability of PC⁃3 cells than neutral medium，whereas BA suppressed the proliferation of PC⁃3 cells in a concentration⁃dependent under acidic microenvironment（P < 0.05）. In addition， the acidic medium also significantly enhanced cell migration，adherence，tumor sphere formation and expressed CD31，CD44 of PC ⁃3 cells than neutral medium. After that，BA was added and the migration，adherence，tumor sphere formation，stem cell property and osteoclast differentiation of PC ⁃ 3 cells were significantly inhibited（P < 0.05）. Conclusion Betulinic acid significantly inhibited the proliferation，migration，adherence，tumor sphere formation，stem cell property and osteoclast differentiation of PC⁃3 cells under acidic microenvironment.