The Ca2+-sensing protein S100A11 of the S100 family is an important mediator of numerous biological functions and pathological conditions including cancer. The receptor for advanced glycation end products (RAGE) has been well accepted as the major receptor for several S100 family members. Here, we take the S100B protein as an antagonist to interfere with the interaction flanked by S100A11 and the RAGE V domain. We employed NMR spectroscopy to describe the interactions between the S100A11 and S100B proteins. 1H-15N heteronuclear single-quantum correlation-NMR titrations showed the potential binding dynamics of S100A11 and S100B interactions. In the HADDOCK program, we constructed the S100A11-S100B heterodimer complex that was then superimposed with the S100A11-S100B complex structure in the same orientation as the S100A11-RAGE V domain complex. This overlay analysis showed that S100B could interfere in the binding section of S100A11 and the RAGE V domain. Additionally, water-soluble tetrazolium-1 assay provided a functional read-out of the effects of these proteins in an in vitro cancer model. Our study establishes that the development of an S100B antagonist could perform a vital part in the treatment of S100- and RAGE-dependent human diseases.