AbstractErbB4 is a member of the ErbB receptor tyrosine kinase family. It has both pro‐ and anti‐oncogenic activities in tumors. Vasculogenic mimicry (VM), a phenomenon in which cancer cells form capillary‐like structures without endothelial cells, has been recognized to be a cause of malignant phenotypes in some solid tumors. Here, we used an in vitro VM formation assay, and demonstrated that ErbB4 negatively regulated VM formation in human breast cancer cells. By using CRISPR/Cas9‐mediated gene knockout, we verified that the depletion of endogenous ErbB4 improved the VM formation capability. Although treatment with neuregulin 1 (NRG1), a ligand of ErbB4, induced the phosphorylation of ErbB4 and promoted VM formation in a dose‐dependent manner, it did not induce such activities in kinase‐dead K751M ErbB4‐overexpressing cells. Moreover, we examined the effect of the missense mutation E872K of ErbB4, which has been reported in multiple tumors, on VM formation, and found that the mutation enhanced the basal phosphorylation level and ErbB4‐mediated VM formation in the absence of NRG1 stimulation. Whereas NRG1 stimulated VM formation, excessive activation of ErbB4 induced a negative effect. In E872K ErbB4‐overexpressing cells, but not in wild‐type ErbB4‐overexpressing cells, the number of VM tubes was significantly decreased by low‐dose treatment with the ErbB inhibitor afatinib. Taken together, our findings demonstrated the significance of ErbB4‐mediated VM formation, and suggested the possibility of ErbB4 mutations as effective targets in breast cancer.