Allostery is an essential biological phenomenon in which perturbation at one site in a biomolecule elicits a functional response at a distal location(s). It is integral to biological processes, such as cellular signaling, metabolism, and transcription regulation. Understanding allostery is also crucial for rational drug discovery. In this work, we focus on an allosteric S100B protein that belongs to the S100 class of EF-hand Ca2+-binding proteins. The Ca2+-binding affinity of S100B is modulated allosterically by TRTK-12 peptide binding 25 Å away from the Ca2+-binding site. We investigated S100B allostery by carrying out nuclear magnetic resonance (NMR) measurements along with microsecond-long molecular dynamics (MD) simulations on S100B/Ca2+ with/without TRTK-12 at different NaCl salt concentrations. NMR HSQC results show that TRTK-12 reorganizes how S100B/Ca2+ responds to different salt concentrations at both orthosteric and allosteric sites. The MD data suggest that TRTK-12 breaks the dynamic aromatic and hydrogen-bond interactions (not observed in X-ray crystallographic structures) between the hinge/helix and Ca2+-binding EF-hand loop of the two subunits in the homodimeric protein. This triggers rearrangement in the protein network architectures and leads to allosteric communication. Finally, computational studies of S100B at distinct ionic strengths suggest that ligand-bound species are more robust to the changing environment relative to the S100B/Ca2+ complex.