ABSTRACT The establishment of epithelial cell polarity requires the assembly of multiprotein complexes and is crucial during epithelial morphogenesis. Three scaffolding proteins, Dlgl, MPP7, and Mals3, can be assembled to form a complex that functions in the establishment and maintenance of apicobasal polarity in epithelial tissues through their L27 domains. Here we report the crystal structure of a 4‐L27‐domain complex derived from the human tripartite complex Dlg1‐MPP7‐Mals3 in combination with paramagnetic relaxation enhancement measurements. The heterotri‐mer consists of 2 pairs of heterodimeric L27 domains. These 2 dimers are asymmetric due to the large difference between the N‐ and C‐terminal tandem L27 domain of MPP7. Structural analysis combined with biochemical experiments further reveals that the loop αA‐αB and helix αB of the C‐terminal L27 domain of MPP7 play a critical role in assembling the entire tripartite complex, suggesting a synergistic tandem L27‐mediated assembling event.—Yang, X., Xie, X., Chen, L., Zhou, H., Wang, Z., Zhao, W., Tian, R., Zhang, R., Tian, C., Long, J., Shen, Y. Structural basis for tandem L27 domain‐mediated polymerization. FASEB J. 24, 4806–4815 (2010). www.fasebj.org