Mapping the conformational pathway of biomolecules is a great challenge because of the large size and complexity of biomolecules. The nudged elastic band (NEB) method has been applied to study the reaction pathways for both small organic molecules and small peptides of a few amino acids. In this work, for the first time, the NEB method was employed to study the conformational pathways of Annexin A1, a membrane-binding protein of 334 amino acids. The N-terminal domain conformational change from the buried state within the core domain to the exposed state outside the core domain is a vital step for Annexin A1 to interact with membranes or target proteins. In this work, multiple molecular dynamics simulations using the NEB method were performed to simulate the N-terminal domain conformational pathway of Annexin A1. Our results suggested that the N-terminal domain of Annexin A1 is removed from the repeat III of the core domain in a sliding motion. The loop region of repeat III covering the N-terminal helix in the buried state does not lift up for the N-terminal to swing out of the pocket; instead, the N-terminal pulls out from the bottom of the core domain. The N-terminal domain linker region (S27-N42) flexibility is critical for the N-terminal domain conformational changes. Our results also suggested a two-step folding process for the helix D in repeat III, M247-V250 folds first followed by the folding of L251-E254. The results demonstrated that the NEB method could be an effective tool for theoretical studies on conformational pathways of biomolecules.