BACKGROUND AND PURPOSE Cystine‐knot miniproteins are characterized by a similar molecular structure. Some cystine‐knot miniproteins display therapeutically useful biological activities, as antithrombotic agents or tumour growth inhibitors. A critical event in the progression of tumours is the formation of new blood vessels. The aim of this work was to test two tomato cystine‐knot miniproteins for their effects on endothelial cell proliferation and angiogenesis in vitro. EXPERIMENTAL APPROACH Two tomato cystine‐knot miniproteins (TCMPs) were expressed and purified either as recombinant or as native proteins from tomato fruits. The Matrigel assay was used to investigate the effects of TCMPs on in vitro angiogenesis. Viability and proliferation of endothelial cells were tested. Extracellular signal‐regulated kinase (ERK)1/2 phosphorylation was assayed in either HUVEC or A431 epidermal growth factor receptor (EGFR)‐overexpressing cells treated with TCMPs. EGFR phosphorylation was tested in A431 cells. KEY RESULTS Both recombinant and native TCMPs inhibited in vitro angiogenesis of HUVEC cells at concentrations of 15–100 nM. The anti‐angiogenic effect of TCMPs was associated with the inhibition of ERK phosphorylation. The two miniproteins did not alter the viability and proliferation of the endothelial cells. CONCLUSIONS AND IMPLICATIONS The anti‐angiogenetic properties of TCMPs are of potential pharmacological interest because they are common and natural components of the human diet, they possess low toxicity, they are active at submicromolar concentrations, they share a common molecular structure that can be used as a molecular platform for the design of molecules with enhanced biological activity.