Fatty acids (FAs) are one of the essential energy sources for physiological processes, and they play a vital role in regulating immune and inflammatory responses, promoting cell differentiation and apoptosis, and inhibiting tumor growth. These functions are carried out by FA binding proteins (FABPs) that recognize and transport FAs. Although the crystal structure of the FA-FABPs complex has long been characterized, the mechanism behind FA binding and dissociation from FABP remains unclear. This study employed conventional MD simulations and enhanced sampling technologies to investigate the atomic-scale complexes of heart fatty acid binding proteins and stearic acid (SA). The results revealed two primary pathways for the binding or dissociation of the flexible long-chain ligand, with the orientation of the SA carboxyl head during dissociation determining the chosen path. Conformational changes in the portal region of FABP during the ligand binding/unbinding were found to be trivial, and the overturn of the ″cap″ or the unfolding of the α2 helix was not required. This study resolves the long-standing debate on the binding mechanism of SA with the long-flexible tail to FABP, which significantly improves the understanding of the transport mechanism of FABPs and the development of related therapeutic agents.