IL-21 is the latest member of the ‘common gamma chain’ cytokine family also including IL-2, IL-4, IL-7, IL-9, and IL-15. Due to its pleiotropic functions in promoting the differentiation and effector function of T cells, B cells and NK cells, IL-21 has attracted significant attention as an immunotherapy target in cancer and infection. Although non-human primate studies and clinical trials showing promising results, data suggested a more potent form than recombinant IL-21 is likely required to deliver clinical efficacy. Our group has identified a novel monoclonal antibody to human IL-21 (clone 2P2). 2P2 enhances the activity of IL-21 for about ten folds in both ex vivo assays and in vivo infection models. By solving the crystal structure of IL-21 in complex with the Fab fragment of 2P2, we found 2P2 binds to IL-21 over a loop bridging helixes C and D (C-D loop). This binding, by changing the conformation and surface charge distribution of IL-21, could optimize its topology and stability to fit the interaction with IL-21 receptor. Based on the structural mechanism of this agonistic antibody, we further generated IL-21 derivatives with ten-folds increased potency. This study reveals a novel mechanism to regulate bioactivity of IL-21 and warrants further development to IL-21 agonistic mAb.