AbstractNumerous age‐linked diseases are rooted in protein misfolding; this has motivated the development of small molecules and therapeutic antibodies that target the aggregation of disease‐linked proteins. Here we explore another approach: molecular chaperones with engineerable protein scaffolds such as the ankyrin repeat domain (ARD). We tested the ability of cpSRP43, a small, robust, ATP‐ and cofactor‐independent plant chaperone built from an ARD, to antagonize disease‐linked protein aggregation. cpSRP43 delays the aggregation of multiple proteins including the amyloid beta peptide (Aβ) associated with Alzheimer's disease and α‐synuclein associated with Parkinson's disease. Kinetic modeling and biochemical analyses show that cpSRP43 targets early oligomers during Aβ aggregation, preventing their transition to a self‐propagating nucleus on the fibril surface. Accordingly, cpSRP43 rescued neuronal cells from the toxicity of extracellular Aβ42 aggregates. The substrate‐binding domain of cpSRP43, composed primarily of the ARD, is necessary and sufficient to prevent Aβ42 aggregation and protect cells against Aβ42 toxicity. This work provides an example in which an ARD chaperone non‐native to mammalian cells harbors anti‐amyloidal activity, which may be exploited for bioengineering.