Glucoamylases, containing starch-binding domains (SBD), have a wide range of scientific and industrial applications. Random mutagenesis and DNA shuffling of the gene encoding a starch-binding domain have resulted in only minor improvements in the affinities of the corresponding protein to their ligands, whereas circular permutation of the RoCBM21 substantially improved its binding affinity and selectivity towards longer-chain carbohydrates. For the study reported herein, we used a standard soluble ligand (amylose EX-I) to characterize the functional and structural aspects of circularly permuted RoCBM21 (CP90). Site-directed mutagenesis and the analysis of crystal structure reveal the dimerisation and an altered binding path, which may be responsible for improved affinity and altered selectivity of this newly created starch-binding domain. The functional and structural characterization of CP90 suggests that it has significant potential in industrial applications.