Crotoxin (CTX) is the primary toxin of South American rattlesnake Crotalus durissus terrificus venom. CTX reduces tumour mass, and tumour cell proliferation and these effects seem to involve the formation of new vessels. Angiogenesis has a key role in tumour growth and progression and is regulated by macrophage secretory activity. Herein, the effect of CTX on macrophage secretory activity associated with angiogenesis was investigated in vitro. Thymic endothelial cells (EC) were incubated in the presence of macrophages treated with CTX (12.5 nM) or supernatants of CTX-treated macrophages and endothelial cell proliferation, migration and adhesion activities, and the capillary-like tube formation in the matrigel-3D matrix was measured. Angiogenic mediators (MMP-2, VEGF and TNF-α) were measured in the cell culture medium. Macrophages pre-treated with CTX and supernatant of CTX-treated macrophages inhibited EC proliferation, adhesion to its natural ligands, and migration (as evaluated in a wound-healing model and Time Lapse assay) activities. Decreased capillary-like tube formation and MMP-2, VEGF and TNF-α levels in the supernatant of macrophages treated with CTX was also described. CTX promotes macrophage reprogramming towards an antiangiogenic phenotype.