MicroRNAs (miRNAs) are involved in tumorigenecity by regulating specific oncogenes and tumor suppressor genes, and their roles in breast cancer stem cells (BCSCs) are becoming apparent. Distinct from the CD44+/CD24−/low sub-population, we have isolated a novel PROCR+/ESA+ BCSC sub-population. To explore miRNA-regulatory mechanisms in this sub-population, we performed miRNA expression profiling and found miR-495 as the most highly upegulated miRNA in PROCR+/ESA+ cells. Coincidently, high upregulation of miR-495 was also found in CD44+/CD24−/low BCSCs, reflecting its potential importance in maintaining common BCSC properties. Ectopic expression of miR-495 in breast cancer cells promoted their colony formation in vitro and tumorigenesis in mice. miR-495 directly suppressed E-cadherin expression to promote cell invasion and inhibited REDD1 expression to enhance cell proliferation in hypoxia through post-transcriptional mechanism. miR-495 expression was directly modulated by transcription factor E12/E47, which itself is highly expressed in BCSCs. These findings reveal a novel regulatory pathway centered on miR-495 that contributes to BCSC properties and hypoxia resistance.