Immunoregulatory protein B7-H3 is involved in the oncogenic and metastatic potential of cancer cells, as well as in drug resistance. Resistance to conventional chemotherapy is an important aspect of melanoma treatment, and a better understanding of how B7-H3 enhances drug resistance may lead to the development of more effective therapies. We investigated the in vitro and in vivo sensitivity of chemotherapeutic agents dacarbazine (DTIC) and cisplatin in sensitive and drug resistant melanoma cells with knockdown expression of B7-H3. We found that knockdown of B7-H3 increased in vitro and in vivo sensitivity of melanoma cells to the chemotherapeutic agents dacarbazine (DTIC) and cisplatin, in parallel with a decrease in p38 MAPK phosphorylation. Importantly, in B7-H3 knockdown cells we observed an increase in the expression of dual-specific MAP kinase phosphatase (MKP) DUSP10, a MKP known to dephosphorylate and inactivate p38 MAPK. DUSP10 knockdown by siRNA resulted in a reversion of the increased DTIC-sensitivity seen in B7-H3 knockdown cells. Our findings highlight the potential therapeutic benefit of combining chemotherapy with B7-H3 inhibition, and indicate that B7-H3 mediated chemoresistance in melanoma cells is driven through a mechanism involving DUSP10-mediated inactivation of p38 MAPK.