Significance Tetraspanin protein (Tspan6) is a member of the tetraspanin family. Using a combination of in vitro and in vivo assays, we demonstrate that Tspan6 functions as a tumor suppressor in colorectal cancer (CRC) by attenuating the epidermal growth factor receptor (EGFR)–based signaling axis. Tspan6 forms a tripartite complex with transmembrane form of TGF-α and an adaptor protein syntenin-1 and negatively regulates secretion of TGF-α. The expression of Tspan6 is frequently decreased in CRC, and this correlates with poor survival. Importantly, the expression of Tspan6 in CRC correlated independently of tumor molecular profile with better patient responses to Cetuximab, an EGFR-targeted therapy. These results identify Tspan6 as a regulator of CRC development and a potential predictive biomarker for EGFR-targeted therapies.