Abstract Oxygen is an essential regulator of cellular metabolism, survival, and proliferation. Cellular responses to oxygen levels are monitored, in part, by the transcriptional activity of the hypoxia inducible factors (HIFs). The existence of hypoxic microenvironment is a common occurrence in many solid tumors including prostate. Previous studies from our laboratories have shown that in human prostate cancer models, cancer stem cells expressing the embryonic stem cell markers SOX2 and OCT3/4 were highly invasive and, importantly, were capable of altering their E-cadherin expression during the process of invasion. In contrast, non-tumorigenic cells which minimally express SOX2 or OCT3/4 were found to be poorly invasive. Furthermore, targeted knockdown of SOX2 or OCT3/4 markedly suppressed the invasion of prostate cancer cells, suggesting an indispensible role of these two stem cell markers in invasiveness. In the present study, we examined the impact of oxygen deprivation on cell invasiveness and stemness in both stem cell enriched and non-stem cell populations in PC3 and DU145 prostate cancer cells. Moreover, the impact of Notch signaling, which has been shown as a critical regulator of prostate cancer development, progression and metastasis, was examined. When cells were exposed to 1% oxygen for 24 h, the invasive capacity of both the stem- and non-stem cell subpopulations increased. Interestingly the non-stem cell subpopulation showed the most striking increase (> 10-fold) in invasion under hypoxic conditions. The expression of several important stem cell factors, including OCT3/4, SOX2, Nanog and c-Myc, also was upregulated under hypoxia in both subpopulations. In addition, the expression of Notch1, Notch2 and their target genes Hey1, Hey2 and HeyL were increased in both subpopulations that had experienced oxygen deprivation (1% O2, 24 h). These findings suggest that although cancer stem cells per se exhibit significantly greater invasive capacity, aberrant microenvironmental conditions such as hypoxia may dramatically enhance the ability of non-stem cells to invade. Consequently, both the stem and non-stem cell subpopulations of tumors may be potential targets for the development of suitable therapeutic interventions designed to inhibit prostate cancer cell invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 485. doi:10.1158/1538-7445.AM2011-485