The amyloid fibril of a fragment of the substrate binding site of αA-crystallin (αAC(71-88)) exhibited chaperone-like activity by suppressing the aggregation of alcohol dehydrogenase (ADH) and luciferase. By contrast, the amyloid fibril of the cytotoxic fragment of amyloid β protein (Aβ(25-35)) facilitated the aggregation of the same proteins. We have determined the zeta potential of the amyloid fibril by measuring their electrophoretic mobility to study the effects of the surface charge on the modulation of protein aggregation. The αAC(71-88) amyloid possesses a large negative zeta potential value which is unaffected by the binding of the negatively charged ADH, indicating that the αAC(71-88) amyloid is stable as a colloidal dispersion. By contrast, the Aβ(25-35) amyloid possesses a low zeta potential value, which was significantly reduced with the binding of the negatively charged ADH. The canceling of the surface charge of the amyloid fibril upon substrate binding reduces colloidal stability and thereby facilitates protein aggregation. These results indicate that one of the key factors determining whether amyloid fibrils display chaperone-like or antichaperone activity is their electrostatic interaction with the substrate. The surface of the αAC(71-88) amyloid comprises a hydrophobic environment, and the chaperone-like activity of the αAC(71-88) amyloid is best explained by the reversible substrate binding driven by hydrophobic interactions. On the basis of these findings, we designed variants of amyloid fibrils of αAC(71-88) that prevent protein aggregation associated with neurodegenerative disorders.