Streptococcal protein G (SpG) is a bacterial surface protein, binding mainly to the Fc (high affinity) and Fab (low affinity) regions of immunoglobulins. The SpG-immobilized affinity chromatography resin is useful for the purification of antibodies. Improvement of the affinity of SpG to Fab enhances its application to the purification of Fab-based antibody fragments. We found four important mutations of SpG, improving its affinity for Fab through affinity maturation using a ribosomal display system. In this article, we overview the calorimetric analyses of these mutations on the function and stability of SpG as an affinity ligand. Isothermal titration calorimetry analysis elucidated the quantitative enthalpic/entropic contributions of these individual mutations on the interaction of SpG with Fab. Differential scanning calorimetry analysis revealed the destabilizing effects of these mutations on the thermodynamic stability of SpG. These calorimetric analyses may contribute to the current understanding of the mechanism of action of such mutations and further refinement of affinity ligands through protein engineering. Additionally, we discuss key features of our new SpGimmobilized affinity chromatography resin such as binding ability and purity profile of target molecules.