In breast cancer, elevated levels of Rab31 in primary tumour tissue are associated with poor patient prognosis. Rab31 is a member of the large Rab protein family of the Ras superfamily of small GTPases. It regulates membrane traffic between the Golgi/trans-Golgi network and the plasma membrane and/or endosomes. In breast cancer cells, increased Rab31 expression enhances cell proliferation, while knock down of Rab31 mRNA levels by short hairpin RNA interference lowers cell proliferation rates. Additionally, increased expression of Rab31 leads to reduced adhesion of cells towards extracellular matrix proteins and decreased invasive capacity through Matrigel. As a control, a Rab31 mutant unable to insert into the Golgi membrane, due to deletion of the two C-terminal cysteine residues (Rab31-ΔCC), was overexpressed in breast cancer cells as well. In contrast to wild-type Rab31, overexpression of the functionally inactive mutant Rab31-ΔCC does not affect in vitro proliferation, adhesion, or invasion. Using microarray analyses and subsequent qPCR, Western blot and/or TGF-β activity assays for validation, Rab31 overexpression in breast cancer cells was demonstrated to modulate expression of other tumour biologically relevant genes, especially genes of the TGF-β superfamily including TGFB1, TGFB2, BMP7, SMAD6, FAS, and TNFSF10. Among others, Rab31 overexpression was found to strongly repress TGF-β, a known suppressor of breast cancer cell proliferation. Thus, Rab31 – depending on its expression level – may represent a major player in the change of the cell biological phenotype of breast cancer cells, i.e. a switch between a proliferative versus invasive phenotype, by mainly affecting TGF-β signalling.