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Overexpression of S100B is routinely used for disease-staging and for determining prognostic outcomes in patients with malignant melanoma. Intracellular interactions between S100B and wild-type (WT)-p53 have been demonstrated to limit the availability of free WT-p53 in tumor cells, inhibiting the apoptotic signaling cascade. Herein, we demonstrate that, while oncogenic overexpression of <i>S100B</i> is poorly correlated (R < 0.3; <i>p</i> > 0.05) to alterations in <i>S100B</i> copy number or DNA methylation in primary patient samples, the transcriptional start site and upstream promoter of the gene are epigenetically primed in melanoma cells with predicted enrichment of activating transcription factors. Considering the regulatory role of activating transcription factors in <i>S100B</i> upregulation in melanoma, we stably suppressed <i>S100b</i> (murine ortholog) by using a catalytically inactive Cas9 (dCas9) fused to a transcriptional repressor, Krüppel-associated box (KRAB). Selective combination of <i>S100b</i>-specific single-guide RNAs and the dCas9-KRAB fusion significantly suppressed expression of <i>S100b</i> in murine B16 melanoma cells without noticeable off-target effects. <i>S100b</i> suppression resulted in recovery of intracellular WT-p53 and p21 levels and concomitant induction of apoptotic signaling. Expression levels of apoptogenic factors (i.e., apoptosis-inducing factor, caspase-3, and poly-ADP ribose polymerase) were altered in response to <i>S100b</i> suppression. <i>S100b</i>-suppressed cells also showed reduced cell viability and increased susceptibility to the chemotherapeutic agents, cisplatin and tunicamycin. Targeted suppression of <i>S100b</i> therefore offers a therapeutic vulnerability to overcome drug resistance in melanoma.