The plasma membrane microdomains, lipid rafts, are involved in regulation of cellular functions such as cell survival and adhesion. Cholesterol is a critical component of lipid rafts in terms of their integrity and functions and rafts disruption by cholesterol depletion can induce detachment-induced cell death. Hypoxia inducible factor-1 (HIF-1) alpha is stabilized in hypoxia and transactivates numerous genes required for cellular adaptation to hypoxia. It is also induced by non-hypoxic stimuli and contributes to cell survival. Because hypoxia inhibits cholesterol synthesis and HIF-1alpha plays a role in this process, we here explored a possible connection between lipid rafts and HIF-1alpha. We investigated whether HIF-1alpha is regulated during cholesterol depletion/rafts disruption in A431 cells in normoxic conditions. Methyl-beta cyclodextrin (MbetaCD), which induces cholesterol depletion, upregulated HIF-1alpha even under normoxic conditions and this upregulation required epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase 1 and 2 activation, but not Akt activation. MbetaCD treatment induced HIF-1alpha upregulation at both the transcriptional and translational levels but not at the posttranslational levels. In addition, MbetaCD robustly induced vascular endothelial growth factor production and stimulated an hypoxia response element-driven luciferase reporter activity under normoxic conditions, indicating that MbetaCD-induced HIF-1alpha is functionally activated. Both EGFR activity and HIF-1alpha expression were higher in the attached cells than in the detached cells after MbetaCD treatment. Furthermore, inhibition of HIF-1alpha by RNA interference accelerated cell detachment, thus increasing cell death, indicating that HIF-1alpha expression attenuates MbetaCD-induced anoikis-like cell death. These data suggest that, depending on cholesterol levels, lipid rafts or membrane fluidity are probably to regulate HIF-1alpha expression in normoxia by modulating rafts protein activities such as EGFR, and this connection between lipid rafts and HIF-1alpha regulation may provide cell survival under membrane-disturbing stress.