Hyperthermia (HT) associated with radiotherapy or chemotherapy is a promising method for cancer treatment, although the molecular mechanisms of this process are not well understood. HT exhibits various antitumor effects, including damage of tumor vasculature. Here, we investigate the effect of HT on in vitro and in vivo angiogenesis. We show that heat treatment of endothelial cells (ECs) affect their differentiation into capillary-like structures in two models of in vitro angiogenesis. Furthermore, the formation of new vessels promoted by angiogenic inducers in the chick embryo chorioallantoic membrane assay is impaired after heat treatment. These effects cannot be explained by direct cytotoxicity but are dependent on modulation of angiogenesis-involved genes. Gene expression profile of ECs subjected to heat shock demonstrates that plasminogen activator inhibitor 1 (PAI-1), a protein involved in the control of extracellular matrix degradation, is specifically up-regulated. The use of anti-PAI-1-neutralizing antibodies reverts the effect of HT on the in vitro EC morphogenesis and in vivo vessel formation. Moreover, microvessel outgrowth from PAI-1(-/-) aortic rings was not affected by HT compared with aortic rings from PAI-1(+/+) mice. Heat treatment of murine mammary adenocarcinomas results in inhibition of tumor growth, associated with a reduction of microvessel number and an increase of PAI-1 expression. These results indicate that heat-mediated PAI-1 induction is an important pathway by which HT exerts its antitumor activity and may represent a rationale for a combined cancer therapy based on HT associated with antiangiogenic molecules.