The LIM domain and tumour suppressor protein Testin (Tes) is downregulated in a variety of human tumours and tumour cell lines. Depending on its conformation, Tes localises to stress fibres and focal adhesions where it forms protein complexes with other members of the actin cytoskeleton, such as zyxin and VASP, and thereby influences cellular processes like cell migration, adhesion or spreading. Tes is a modular protein and Tes variants lacking specific domains, localize to different locations in cells. To better understand the molecular basis of its function, we utilized an interaction proteomics approach combined with pathway analysis. This revealed proteins present in complexes in which Tes participates as a function of its modular structure as well as novel Tes interaction partners. We demonstrate that Tes interacts with a short isoform of the glucocorticoid receptor (GR), independently of the conventional full length GRα, a transcription factor important in regulating cellular metabolism and immune function. Tes also interacts with the focal adhesion protein and GR-coactivator Hic-5. In addition, we found that upon overexpression, Tes and Hic-5 induce opposite effects on cell spreading on a fibronectin matrix.