S100B is a calcium sensing protein with intracellular and extracellular roles. It is one of the EF hand homodimeric protein of the S100 protein family, which is known to interact with various protein targets. Extracellular S100B has been recently reported to interact with FGF2 in RAGE receptor independent manner. Residue level information of S100B interaction with FGF2 would be useful to understand the varied target recognition ability of S100B and can further explain its role in effecting extracellular signaling diversity. This is the first report which details the elucidation of structural level interactions between human S100B and FGF2. The S100B-FGF2 interface was mapped by heteronuclear NMR spectroscopic studies. The binding affinity between S100B and FGF2 is modestly strong (Kd ~10.0 μM), as determined by thermodynamic association studies. We utilized the NMR experiment driven HADDOCK docking approach for determining the structure of the S100B-FGF2 complex that is further proved stable using unrestrained Molecular dynamics (MD) simulations. We pronounced the interface regions between S100B and FGF2 in the resolved complex. Mechanistic insights into the S100B-FGF2 complex interface led us to propose the novel role of S100B in FGF2 mediated FGFR1 receptor inactivation. Functional assay and site directed mutagenesis studies provided the necessary evidence to support this hypothesis.