Myeloid leukaemia factor 1 (MLF1) binds to 14-3-3 adapter proteins by a sequence surrounding Ser34 with the functional consequences of this interaction largely unknown. We present here the high-resolution crystal structure of this binding motif [MLF1(29-42)pSer34] in complex with 14-3-3e and analyse the interaction with isothermal titration calorimetry. Fragment-based ligand discovery employing crystals of the binary 14-3-3e/MLF1(29-42)pSer34 complex was used to identify a molecule that binds to the interface rim of the two proteins, potentially representing the starting point for the development of a small molecule that stabilizes the MLF1/14-3-3 protein-protein interaction. Such a compound might be used as a chemical biology tool to further analyse the 14-3-3/MLF1 interaction without the use of genetic methods. Database Structural data are available in the Protein Data Bank under the accession number(s) 3UAL [14-3-3e/MLF1(29-42)pSer34 complex] and 3UBW [14-3-3e/MLF1(29-42)pSer34/3-pyrrolidinol complex] Structured digital abstract • 14-3-3 epsilon and MLF1 bind by x-ray crystallography (View interaction) • 14-3-3 epsilon and MLF1 bind by isothermal titration calorimetry (View Interaction: 1, 2).