The Homeobox B9 (HOXB9) is a homeodomain-containing transcription factor that participates in the progressionof various malignancies. Nevertheless, the functional role of HOXB9 in prostate cancer cells is largelyunknown. Hence, we aimed to address the effect of HOXB9 on the progression of prostate cancer cells. Smallinterfering RNA (siRNA) against HOXB9 was used to downregulate HOXB9 expression in PC3 and DU145cells. Western blotting was performed to detect the expression levels of HOXB9 and other related proteins. Cellproliferation was tested by the Cell Counting Kit-8 (CCK-8) and cell cycle and apoptosis were investigated byflow cytometry. Angiogenesis was examined using tube formation assays The Transwell assays were carriedout to assess the migratory and invasive capacities of cells. Here, we found that HOXB9 knockdown significantlyreduced cell proliferation via inducing cell cycle arrest at G1 phase. This treatment also reducedangiogenesis, migration and invasion abilities of PC3 and DU145 cells in vitro. We also found that HOXB9knockdown inhibits the activation of the PI3K/AKT signaling pathway in prostate cancer cells. In conclusion,our findings revealed that HOXB9 promotes prostate cancer progression and might be a novel and effectivetherapeutic target for human prostate cancer.