We recently established the critical role of the PTEN/MAGI‐lb signalosome in stabilization of cell‐cell contacts and suppression of invasiveness. The PTEN tumor suppressor is recruited to E‐cadherin junctional complexes through the binding to the second PDZ domain of the MAGI‐1b scaffolding molecule, whereas β‐catenin interacts with the fifth PDZ domain. To identify additional effectors of this signalosome, we used yeast 2‐hybrid screening. Among the clones identified, we focused on TRIP6, which belongs to the zyxin family of proteins. We demonstrated that TRIP6 interacted directly with MAGI‐lb by binding to its fifth PDZ domain. Ectopic expression of TRIP6 induced invasiveness in the epithelial MDCK and MDCKts‐src cells in a PI3‐kinase‐ and a NF‐κB‐dependent manner and impaired cell‐cell aggregation at least in part by uncoupling adherens junctional complexes from the cytoskeleton. The TRIP6Stop473 mutant, which lacks the PDZ binding motif, was still able to increase NF‐κB and Akt activities but did not promote invasiveness or interfere with cell‐cell aggregation. Intracellular delivery of competing peptides corresponding to TRIP6 or β‐catenin C terminus restored invasive properties in MDCKts‐src TRIP6Stop473 cells, highlighting the requirement of PDZ scaffolds in junctional complexes activity. TRIP6 overexpression in colon tumors suggest its critical role in cancer progression.— Chastre, E., Abdessamad, M., Kruglov, A., Bruyneel, E., Bracke, M., Di Gioia, Y., Beckerle, M. C., vanRoy, F., Kotelevets, L. TRIP6, a novel molecular partner of the MAGI‐1 scaffolding molecule, promotes invasiveness. FASEB J. 23, 916–928 (2009)