Glycosaminoglycans (GAGs) are long unbranched anionic polysaccharides made up of repetitive disaccharide units involved in biologically relevant processes in the extracellular matrix such as cell proliferation and communication. A GAG can be bound in antiparallel energetically comparable orientations on the protein surface, and these orientations are, therefore, difficult to distinguish both experimentally and computationally. In this study, for the first time we analyzed the impact of the GAG chain polarity on the interactions with Fibroblast Growth Factors-1 and -2 (FGF-1 and FGF-2). We performed a series of 1 μs molecular dynamics simulations of the FGF-1 and FGF-2 complexes with heparin (HP), a GAG representative, of different length. We analyzed the relationship between the HP orientation, energetic, and conformational space characteristics of FGF-1-HP and FGF-2-HP complexes. We concluded that HP can be bound by these proteins in the same binding sites but in different orientations, while the orientation present in the experimental structure might be favorable. Our data presented in this study provide a novel view on the impact of GAG polarity on the specificity of protein-GAG complex formation, which is an essential aspect for the proper understanding of the intermolecular interactions in these systems.