Cancer stem cells (CSCs), or tumor-initiating cells, are involved in tumor progression and metastasis. MicroRNAs (miRNAs) regulate both normal stem cells and CSCs^,,,, and dysregulation of miRNAs has been implicated in tumorigenesis. CSCs in many tumors—including cancers of the breast, pancreas, head and neck, colon^,, small intestine, liver, stomach, bladder and ovary—have been identified using the adhesion molecule CD44, either individually or in combination with other marker(s). Prostate CSCs with enhanced clonogenic and tumor-initiating and metastatic^, capacities are enriched in the CD44⁺ cell population, but whether miRNAs regulate CD44⁺ prostate cancer cells and prostate cancer metastasis remains unclear. Here we show, through expression analysis, that miR-34a, a p53 target^,,,,, was underexpressed in CD44⁺ prostate cancer cells purified from xenograft and primary tumors. Enforced expression of miR-34a in bulk or purified CD44⁺ prostate cancer cells inhibited clonogenic expansion, tumor regeneration, and metastasis. In contrast, expression of miR-34a antagomirs in CD44⁻ prostate cancer cells promoted tumor development and metastasis. Systemically delivered miR-34a inhibited prostate cancer metastasis and extended survival of tumor-bearing mice. We identified and validated CD44 as a direct and functional target of miR-34a and found that CD44 knockdown phenocopied miR-34a overexpression in inhibiting prostate cancer regeneration and metastasis. Our study shows that miR-34a is a key negative regulator of CD44⁺ prostate cancer cells and establishes a strong rationale for developing miR-34a as a novel therapeutic agent against prostate CSCs.