Selenoprotein P (SeP) is synthesized mainly by the liver and functions as a selenium (Se)-supply protein to maintain Se levels in peripheral tissue. SeP is identified as a hepatokine, promoting insulin resistance in type 2 diabetes. Recently, we found that excess SeP significantly decreased in pancreas insulin levels and high glucose-induced insulin secretion. Immunohistochemical analysis of pancreatic islets showed a reduction of the size of the islets in SeP-treated mice. Further, the islets of SeP-treated mice showed a disturbed morphology with irregular shape and a concomitant decrease of α cells with β cells. Impaired effects of excess SeP on insulin secretion were also observed in isolated islets and a MIN6 cell model of pancreatic β cells. We identified anti-human SeP monoclonal antibody (Ab) AE2 as neutralizing Ab with activity against Se-supply of SeP. Administration of AE2 significantly improved insulin levels and high glucose-induced insulin secretion in vitro and in vivo. Neutralizing Ab of mouse SeP improved insulin secretion in a mouse model of diabetes, such as KKAy mice and mice fed with a high-fat, high-sucrose diet. These results suggest the critical role of SeP levels in function of pancreatic β cell. This report describes a novel molecular strategy for the development of type 2 diabetes therapeutics targeting SeP.