- The ErbB family of tyrosine kinase receptors is a key element in preserving cell growth homeostasis. This family is comprised of four single-transmembrane domain proteins designated ErbB-1–4. Ligand binding initiates dimerization followed by tyrosine phosphorylation and signaling, which when uncontrolled lead to cancer. Accordingly, extensive research has been devoted to finding ErbB-intercepting agents, directed against ErbB-1 and ErbB-2, but so far, no inhibitor has targeted the transmembrane domain (TMD), which is instrumental for receptor dimerization and activation. Moreover, no antitumor agents targeted ErbB-3, which although it cannot generate signals in isolation, its heterodimerization with ErbB-2 leads to the most powerful and oncogenic signaling unit in the ErbB family. Here, to further elucidate the role of the interactions between the TMDs of the ErbB family in cancer, we investigated peptides derived from the TMDs of ErbB-1 and ErbB-2. We then focused on the C-terminal domains (B2C) and their analogue, named B2C-D, that contains both d- and l-amino acids. Both peptides incorporated the distal GXXXG dimerization motif to target the TMD of ErbB-3. Our results revealed that B2C-D is highly active both in vitro and in vivo. It significantly inhibits neuregulin- and EGF-induced ErbB activation, impedes the proliferation of a battery of human cancer cell lines, and retards tumor growth in vivo. Notably, combining low concentrations of B2C-D and gemcitabine chemotherapy completely arrested proliferation of pancreatic cancer cells. Biochemical and in vitro interaction studies suggest direct interference with the assembly of the wild-type ErbB-2–ErbB-3 heterodimer as the underlying mode of action. To the best of our knowledge, this is the first agent to target the TMDs of ErbB to delay tumor growth and signaling.