Signet-ring cell carcinoma is one of the most malignant tumors, classified histologically as a poorly differentiated adenocarcinoma. The ErbB2/ErbB3 complex is often constitutively activated, which suggests that the ErbB2/ErbB3 signaling pathway may be important for malignancy of this tumor. However, the mechanism underlying this activation has not been understood. Here, we show that ErbB2 and Muc4 bind in signet ring carcinoma cells, which was not seen in highly differentiated adenocarcinoma cell lines. ErbB3 was suggested to be a substrate of ErbB2 because knockdown of ErbB2 resulted in less phosphorylation of ErbB3. Inhibition of expression of Muc4 at the cell surface by the treatment of the cells with benzyl-GalNac, an inhibitor of mucin secretion, blocked phosphorylation of ErbB3, suggesting that activity of ErbB2 depends on the expression of Muc4. These results supply the biochemical backgrounds in recent studies suggesting the contribution of Muc4 in the tumorigenesis.