(1) Vascular endothelial growth factor (VEGF) is a potent angiogenic factor. It has been recently suggested that the inducible heme oxygenase (HO-1) isoform may play a role in angiogenesis. (2) The aims of this study were to determine, in chicken embryo chorioallantoic membranes (CAM), whether VEGF increases HO-1 protein expression, and, if so, by which molecular mechanism, and whether HO-1 activity is required for VEGF-induced angiogenesis. (3) Treatment of CAMs with VEGF for 48 h caused a significant increase in HO-1 protein expression, simultaneously with angiogenesis. (4) VEGF-stimulated angiogenesis in CAMs was markedly attenuated by the HO inhibitor zinc mesoporphyrin (ZnMP). This inhibitory effect of ZnMP was not observed with copper mesoporphyrin (CuMP), a metalloporphyrin that has a similar structure to ZnMP but does not inhibit HO enzymatic activity. (5) Overexpression of HO-1 protein elicited by VEGF in CAMs was significantly attenuated by the intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM). The effects of BAPTA-AM were, in turn, compensated by the calcium ionophore A-23187. (6) In addition, the protein kinase C inhibitor staurosporine significantly attenuated, in a dose-dependent manner, the VEGF-stimulated HO-1 induction observed in CAMs. (7) These results demonstrate, for the first time, that VEGF upregulates HO-1 protein expression in vivo in CAMs by a mechanism dependent on an increase in cytosolic calcium levels and activation of protein kinase C. Our findings also suggest that HO-1 activity is necessary for VEGF-induced angiogenesis in CAMs.