Objective To investigate the effects of Betulinic acid（BA）on the stemness，invasion，migra⁃ tion，adhesion and hypoxia inducible factor⁃1α（HIF⁃1α）expression in human prostate cancer bone metastatic PC⁃3 cells under hypoxia. Methods PC⁃3 cells were treated with various concentrations of BA solution，the cell viability was detected by CCK8 and the IC50 value of 50% inhibition concentration was calculated，and then divided into IC50 concentration BA group and control group to detect the effect of betulinic acid on the proliferation of PC⁃3 cells，PC⁃3 cells were treated with normoxic，Hypoxia group and Hypoxia+IC50（10 μmol/L）BA group，and the effects of BA on cell invasion and migration were detected by Transwell assay. The effect of BA on cell adherence was determined using a cell adhesion test，and the ability of PC⁃3 cells to self⁃renew following BA treatment was determined using a tumor spheroidization assay. The effect of BA on the expression of HIF⁃1α，CD31，and CD44 was detected using polymerase chain reaction（PCR）and Western blot immunoblotting，while the effect of BA on the expression of VEGF，IL⁃8，IL⁃1β，IL⁃6，and TNF⁃α factors was detected using an enzyme linked immunosorbent assay（ELISA）. Results BA inhibited the proliferation of PC ⁃3 cells in a concentration ⁃and time ⁃dependent manner，and its IC50 was 10 μmol/L. PC⁃3 cells stemness，invasion，migration，adhesion ability were significantly increased under hypoxia. However，after BA treatment，the aforementioned phenomenon was reversed. Western blot analysis revealed that BA could inhibit the expression of HIF ⁃ 1α protein，CD31 and CD44 protein in PC ⁃3 cells（P < 0.01）。The results of PCR assay revealedthat BA could downregulation the mRNA expression of HIF⁃1αand mRNA of CD31 and CD44（P < 0.01）. The results of ELISA test showed that BA could inhibit the expression of VEGF，IL⁃8，IL⁃1 β，IL⁃6 and TNF⁃α（P < 0.01）. The results of ELISA test showed that BA could inhibit the expression of VEGF，IL⁃8，IL⁃1 β，IL⁃6 and TNF⁃α（P < 0.01）. Conclusion Under hypoxia，BA inhibits PC⁃3 cell stemness，invasion，migration，and adhesion. It′s possible that BA inhibits the expression of HIF⁃1α，CD31，CD44，and cytokines including VEGF，IL⁃8，IL⁃1β，IL⁃6，and TNF⁃α.