Previously, we cloned Heat shock protein A12B (HspA12B), the newest member of a recently defined subfamily of proteins distantly related to the Hsp70 family that are enriched in atherosclerotic lesions. We have found that HspA12B is predominantly expressed in vascular endothelium, and that it is involved in angiogenesis which we probed by in vitro angiogenesis assays (Matrigel), migration assays and Directed In Vivo Angiogenesis Assay (DIVAA). Hsp70s are molecular chaperones that are inducible by stress and have been found to be anti-apoptotic (Li et al. 2000; Nylandsted et al. 2000; Garrido et al. 2001). Because of its homology to Hsp70, we propose that it is the first endothelial-specific chaperone that is required for angiogenesis and interacts with known angiogenesis regulators. To begin to understand the molecular mechanisms underlying the role of HspA12B in angiogenesis, we turned our attention to identifying proteins that are involved in angiogenesis and also interact with HspA12B. Through the use of a yeast two-hybrid (Y2H) system HspA12B was found to interact with a known angiogenesis regulator, A Kinase Anchoring Protein 12 (AKAP12). This interaction was confirmed by co-immunoprecipitation and by colocalization. In primary human umbilical vein endothelial cells (HUVECs), shRNA mediated HspA12B knockdown increased AKAP12 levels and decreased VEGF by more than 75%, whereas HspA12B over-expression decreased AKAP12 and more than doubled VEGF levels. We further identified a 32-Amino Acid (32-AA) domain in AKAP12 that mediates interaction with HspA12B. Over-expression of this 32-AA domain in HUVECs disrupted the HspA12B-AKAP12 interaction and decreased VEGF expression suggesting the importance of the HspA12B-AKAP12 interaction in regulating VEGF. This is the first evidence that HspA12B promotes angiogenesis resulting in up-regulation of VEGF by suppressing AKAP12. Consistent with the proposed role in angiogenesis, HspA12B was also found to be increased in endothelial cells (ECs) by angiogenic stresses including hypoxia and shearing stress while knockdown of HspA12B abolished hypoxia-induced tubule formation. This work provides new insight into the mechanisms controlling angiogenesis by providing the first example of an EC-specific molecular chaperone that acts as a regulator of angiogenesis and lays the foundation for future studies of HspA12B-derived therapeutics for angiogenesis related diseases.