CD44 is a multifunctional protein involved in cell adhesion and signaling. The role of CD44 in prostate cancer (PCa) development and progression is controversial with studies showing both tumor-promoting and tumor-inhibiting effects. Most of these studies have used bulk-cultured PCa cells or PCa tissues to carry out correlative or overexpression experiments. The key experiment using prospectively purified cells has not been carried out. Here we use FACS to obtain homogeneous CD44+ and CD44− tumor cell populations from multiple PCa cell cultures as well as four xenograft tumors to compare their in vitro and in vivo tumor-associated properties. Our results reveal that the CD44+ PCa cells are more proliferative, clonogenic, tumorigenic, and metastatic than the isogenic CD44− PCa cells. Subsequent molecular studies demonstrate that the CD44+ PCa cells possess certain intrinsic properties of progenitor cells. First, BrdU pulse-chase experiments reveal that CD44+ cells colocalize with a population of intermediate label-retaining cells. Second, CD44+ PCa cells express higher mRNA levels of several ‘stemness’ genes including Oct-3/4, Bmi, β-catenin, and SMO. Third, CD44+ PCa cells can generate CD44− cells in vitro and in vivo. Fourth, CD44+ PCa cells, which are AR−, can differentiate into AR+ tumor cells. Finally, a very small percentage of CD44+ PCa cells appear to undergo asymmetric cell division in clonal analyses. Altogether, our results suggest that the CD44+ PCa cell population is enriched in tumorigenic and metastatic progenitor cells.