Epithelial tight junctions recruit different types of signalling proteins that regulate cell proliferation and differentiation. Little is known about how such proteins interact functionally and biochemically with each other. Here, we focus on the Y-box transcription factor ZONAB (zonula occludens 1-associated nucleic-acid-binding protein)/DbpA (DNA-binding protein A) and the Rho GTPase activator guanine nucleotide exchange factor (GEF)-H1/Lbc's first cousin, which are two tight-junction-associated signalling proteins that regulate proliferation. Our data show that the two proteins interact and that ZONAB activity is Rho-dependent. Overexpression of GEF-H1 induces accumulation of ZONAB in the nucleus and activates transcription. Microtubule-affinity regulating kinase/partition-defective-1, another type of GEF-H1-associated signalling protein, remains in the cytoplasm and partially co-localizes with the exchange factor. GEF-H1 and ZONAB are required for expression of endogenous cyclin D1, a crucial RhoA signalling target gene, and GEF-H1-stimulated cyclin D1 promoter activity requires ZONAB. Our data thus indicate that GEF-H1 and ZONAB form a signalling module that mediates Rho-regulated cyclin D1 promoter activation and expression.