Fibroblast growth factor 2 (FGF2) regulates signal transduction by forming complexes with its receptors, FGF receptors (FGFRs), and heparan sulfate (HS), playing a crucial role in biological systems. Although HS has been suggested to modulate FGF/FGFR signaling as a co-receptor, multiple hypotheses exist regarding how HS affects FGF/FGFR signaling, and the mechanism remains unclear. In this study, to highlight the role of FGF2/HS interaction in FGF2/FGFR1 signaling, FGF2 mutants with reduced HS-binding affinity were rationally designed through in silico analysis. These FGF2 mutants exhibited reduced HS affinity by more than two orders of magnitude while maintaining binding affinity to FGFR1. In addition, these mutants retained their thermal stability. Cellular assays using the FGF2 mutant suggested that, contrary to previous reports, the contribution of the FGF2/HS interaction in FGF2/FGFR1 signaling may be limited. The mutant FGFs that specifically alter the interaction with HS, achieved in this study, would contribute to an understanding of the role of FGF/HS interaction in FGF/FGFR signaling.