The S100B protein, a member of the S100 protein family, is highly elevated during the progression of melanoma. Elevated level of S100B in the serum is used as a marker in melanoma and other cancers. However, the function of S100B in the progression of melanoma is not completely understood. Here we uncovered a regulatory mechanism that defines interplay between S100B, Interleukin-6 (IL-6), STAT3, and CREB. First, we show that S100B inhibits IL-6 mRNA and protein levels. Silencing S100B in melanoma cells induces the expression and secretion of IL-6, which in turn induced STAT3 phosphorylation and activation. S100B exerts its effect on the IL-6/STAT3 pathway via the p90 ribosomal S6 kinase (RSK), and the phosphorylation and activation of transcription factor CREB. High S100B in melanoma cells binds to RSK and sequesters RSK in the cytoplasm. Silencing of S100B enables RSK nuclear translocation, which in turn elevates CREB phosphorylation and its transcriptional activity in the nucleus to induce IL-6 expression. Therefore, high S100B in melanoma suppresses IL-6 expression. Since IL-6 was shown to inhibit the proliferation of melanocytes and early stage melanoma cells, we propose that the suppression of IL-6 by S100B evolved to circumvent the inhibitory effect of IL-6 and STAT3 at early stages of melanoma. It is also possible that suppression of IL-6 by S100B evolved to curb the local immune response, which otherwise would be elevated by the secreted IL-6. Indeed, we show that S100B silencing upregulates the expression of several chemokine ligands, chemokine receptors, and interleukins that are involved in the immune response. For example, we show that S100B depletion induces expression of cytokines, CSF-1 and CSF-2, in STAT3-dependent manner. These results suggest that S100B inhibits interleukins and chemokines to perhaps curb the immune response within the tumor microenvironment. Future experiments in mice models and cell culture systems are necessary to further evaluate the role of S100B in regulating the immune response in malignant melanoma. We are also in search for S100B inhibitors that can potentially prevent S100B-target complex formation and reduce tumor growth. Cellular characterization of pentamidine/heptamidine derivatives, covalently bound inhibitors, and SC0025 were explored in the non-targeting scrambled and stable S100B knockdown WM115 cell line. We show that lower concentrations of SC124, SBi4172, SC1982, and SC0025 inhibitors were needed to inhibit cell growth in the non-targeting scrambled WM115 versus stable S100B knockdown WM115 cells indicating that these compounds have specificity toward S100B-containing cells. We also show that SC1982 restored p53 protein level by more than 2-fold, while SC0025 restored only IL-6 protein level. The characterization of SC1982 and SC0025, which occupy sites 2 and 3 within S100B binding pockets, respectively, reveals that occupying one binding site within S100B binding pocket is not sufficient to restore multiple S100B targets. The goal is to identify S100B inhibitors that can prevent binding of multiple targets and reduce tumor growth as therapeutic intervention for melanoma therapy with elevated S100B.