Isomerization of L-aspartate (L-Asp) into D-aspartate (D-Asp) occurs naturally in proteins at a rate that is much faster than that of other amino acid types. Accumulation of D-Asp is age-dependent, which could alter protein structures and, therefore, functions. Site-specific introduction of D-Asp can accelerate aggregation kinetics of a variety of proteins associated with misfolding diseases. Here, we showed by thioflavin T fluorescence that the isomerization of L-Asp at different positions of amyloid β peptide 1-42 (Aβ42) generates opposing effects on its aggregation kinetics. We further determined the atomic structures of Aβ42 amyloid fibrils harboring a single D-Asp at position 23 and two D-Asp at positions 7 and 23 by cryo-electron microscopy helical reconstruction - cross-validated by cryo-electron tomography and atomic force microscopy - to reveal how D-Asp