High altitude and many diseases (e.g. cardiovascular diseases, cancer and chronic wounds) are associated with a limited oxygen supply. However, also normal mammalian embryonic development depends on a hypoxic environment. Cells can adapt to hypoxia by various mechanisms. Yet, the effects of hypoxia on cell morphology and the actin cytoskeleton are largely unknown. Here we present a comprehensive examination of the cell architecture and function of L929 fibroblasts (1% O₂). Our results demonstrate that cells cultivated in hypoxia show striking morphological differences compared to cells cultivated under normoxic conditions (20% O₂). These changes include an enlargement of cell size, changing cell protrusions, increased numbers of focal contacts and a loss of cell polarization. Furthermore the ß- and γ -actin distribution is greatly altered. In L929 fibroblasts these hypoxic adjustments in morphology are associated with enhanced cell spreading and a decline of cell motility in wound closure assays and single cell motility assays. The hypoxia inducible factor -1α (HIF-1α) is stabilized in hypoxia and plays a pivotal role in the response to changes in oxygen availability by transcriptional upregulation of a large number of genes. Using a shRNA-approach our results show that the observed cellular hypoxic alterations partly depend on HIF-1α stabilization.