Simple Summary Tetraspanins are cell surface proteins that induce the compartmentalization of molecules with receptor, signaling, or enzymatic properties in microdomains that could serve the integration of transmembrane signals. Several knockouts illustrate their biological functions such as infertility (CD9/CD81), lymphoma development (CD37), immunological deficiency (CD81), or renal insufficiency (CD151). The link between molecular interactions and organ defects remains often difficult to decipher, as is the relationship between the expression of some tetraspanins and the outcome of tumors. High expression of Tspan8 in tumors is associated with poor prognosis, which may be due to increased cell motility and angiogenesis. In a model of colorectal tumor, we explored Tspan8 interacting molecules by mass spectrometry and found that compared to CD9, Tspan8 specifically recruits the endothelin converting enzyme ECE1 to tetraspanin-enriched microdomains and positively modulates its enzymatic activity. Convergence of Tspan8 and endothelin axis in tumor biology offers a new area of research.Abstract Tspan8 is a member of the tetraspanins family of cell surface molecules. The ability of tetraspanins to organize membrane microdomains with other membrane molecules and interfere with their function suggests that they could act as surface integrators of external or internal signals. Among the first identified tetraspanins, Tspan8 promotes tumor progression and metastasis, presumably by stimulating angiogenesis and cell motility. In patients, its expression on digestive tract tumors seems to be associated with a bad prognosis. We showed previously that Tspan8 associates with E-cadherin and EGFR and modulates their effects on cell motility. Using Mass spectrometry and western blot, we found a new partner, the endothelin converting enzyme ECE1, and showed that Tspan8 amplifies its activity of conversion of the endothelin-1 precursor bigET1 to endothelin. This was observed by transduction of the colon carcinoma cell line Isreco1, which does not express Tspan8, and on ileum tissue fragments of tspan8ko mice versus wild type mice. Given these results, Tspan8 appears to be a modulator of the endothelin axis, which could possibly be targeted in case of over-activity of endothelins in biological processes of tissues expressing Tspan8.