The aim of the present study was to investigate the functions of RACK1 and its involvement in mechanisms of prostate cancer (PC) cell proliferation, invasion and metastasis. The proliferation, invasion and metastasis of stably transfected DU145 cells with RACK1 was evaluated in vitro as well as in vivo following the establishment of nude mouse models. The expression of Ki67, RACK1, PTEN and androgen receptor (AR) in PC was detected by immunohistochemical analysis. Our results indicated that RACK1 promotes PC cell proliferation, invasion and metastasis in vitro and in vivo. However, knockdown of RACK1 by siRNA in vitro inhibited PC cell proliferation, migration and invasion. PTEN downregulation and Ki67 upregulation were also altered with the upregulation of RACK1; RACK1 staining was strongly correlated with PTEN downregulation and Ki67 upregulation. These data demonstrated that increased RACK1 expression is important in promoting PC cell proliferation, invasion and metastasis in vitro and in vivo.