The deposition of aggregated human islet amyloid polypeptide (hIAPP) in the pancreas, that has been associated with β-cell dysfunction, is one of the common pathological features of patients with type 2 diabetes (T2D). Therefore, hIAPP aggregation inhibitors hold a promising therapeutic schedule for T2D. Chitosan oligosaccharides (COS) have been reported to exhibit a potential antidiabetic effect, but the function of COS on hIAPP amyloid formation remains elusive. Here, we show that COS inhibited the aggregation of hIAPP and disassembled preformed hIAPP fibrils in a dose-dependent manner by thioflavin T fluorescence assay, circular dichroism spectroscopy, and transmission electron microscope. Furthermore, COS protected mouse β-cells from cytotoxicity of amyloidogenic hIAPP, as well as apoptosis and cycle arrest. There was no direct binding of COS and hIAPP, as revealed by surface plasmon resonance analysis. In addition, both chitin-oligosaccharide and the acetylated monosaccharide of COS and glucosamine had no inhibition effect on hIAPP amyloid formation. It is presumed that, mechanistically, COS regulate hIAPP amyloid formation relating to the positive charge and degree of polymerization. These findings highlight the potential role of COS as inhibitors of hIAPP amyloid formation and provide a new insight into the mechanism of COS against diabetes.