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Annexins (Anxs) are a family of highly homologous proteins that bind and aggregate lipid vesicles in the presence of calcium. All members of the family contain a variable N-terminus determining specific functions, followed by a conserved core region responsible for the general calcium-dependent lipid-binding property. The core structure consists of four homologous domains (D I -D IV ), each consisting of a right-handed super-helix of five α-helices. We present data from a combination of site-directed mutagenesis, NMR, and circular dichroism showing that the G25-D34 region of the N-terminus as well as the contacts between residues D38A, R63A, and Q67A of AnxA2-D I are crucial for the autonomous folding and stability of D I of AnxA2. However, we also show that the folding of the full-length protein is very robust in that mutations and truncations that disrupted the folding of AnxA2-D I did not abolish the folding of full-length AnxA2, only lowering its thermal stability. This robustness of the folding of full-length AnxA2 is likely to be mediated by the existence of at least one transient nonnative intermediate as suggested by our kinetic data using stopped-flow fluorescence experiments. We also show that hydrophobic amino acids in AnxA2-D I involved in interfacial contacts with AnxA2-D IV are important for the cooperative folding and stability of the full-length protein. Mutating all of the V57E-V98R-G101Y residues in AnxA2-D I did not affect the folding of the domain, only its stability, but prevented the cooperative folding of the full-length protein. Our collective results favor a highly cooperative and robust folding process mediated by alternative intermediate steps. Since AnxA2 is a multifunctional protein involved in several steps of the progression of cell transformation, these data on structure and folding pathways are therefore crucial to designing anticancer drugs targeting AnxA2., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Biophysical Society. Published by Elsevier Inc. All rights reserved.)