Interaction between extracellular matrices and cancer cell receptors frequently alters signal transduction pathways, leading to malignant transformation and metastasis. Hyaluronan (HA) is a tumor promoter and enhancer in transformation of androgen-independent (AI) prostate cancer (CaP); however, the signal transduction pathway involved in this mechanism remains unclear. We report here that HA-mediated CD168, a receptor for HA-mediated motility, and its downstream signal molecules, including ROK1, Gab-1, PI3K*p110alpha and eIF4E3, accelerate the progression of AI rather than androgen-dependent CaP and enhance AI cell invasion and metastasis in human bone marrow endothelial layers. MicroRNA-based small hairpin RNA-mediated suppression of ROK1 can reverse the malignant role of CD168 signaling in human AI CaP PC3 and DU145 cells. This differential activation of ROK-PI3K signaling in AI CaP cells may provide clues to shed light on some mechanisms of cancer relapse after androgen ablation. These findings reveal a novel signal transduction mechanism for matrix-mediated cancer transformation and metastasis in hormone-refractory CaP.