Although surgery and radiotherapy have been the standard treatment modalities for head and neck squamous cell carcinoma (HNSCC), the integration of cisplatin (CDDP)-based therapy has led to improvements in local and regional control of disease for patients. However, many trials show that only 10-20% of patients benefit from this treatment intensification, which can result in profound treatment-associated morbidity and mortality. Moreover, the marginal survival improvement suggests that CDDP resistance is an innate characteristic of HNSCC. To elucidate the biological mechanisms underpinning CDDP resistance in HNSCC, we utilized an experimental model of CDDP resistance in this disease. We first observed significant enhancements in local tumor growth and metastasis, as well as adverse survival, in CDDP-resistant (CR) tumors compared with sensitive tumors. To elucidate the molecular mechanisms of this phenotype, we undertook a systems biology-based approach utilizing high-throughput PCR arrays, and we identified a significant suppression of KiSS1 mRNA and protein expression in the CR cells, but no significant regions of genomic loss with array comparative genomic hybridization. Genetic suppression of KiSS1 in CDDP-sensitive cell lines rendered them CR, an observation that was mechanistically linked to alterations in glutathione S-transferase-π expression and function. We next confirmed that, in human HNSCC tumors, loss of KiSS1 expression was associated with metastatic human HNSCC tumors compared with non-metastatic tumors. Genetic reconstitution of KiSS1 in CR cells abrogated cellular migration and induced CDDP sensitivity. To confirm these findings in a murine model, either CR or KiSS1-transfected CR cells were studied in an orthotopic model of HNSCC, or survival studies revealed significant improvement in survival of the mice bearing CR-KiSS1 tumors. Mechanistically, alterations in apoptotic pathways and CDDP metabolism contributed to KiSS1-associated chemotherapy sensitization. These studies provided further direct evidence for the role of KiSS1 loss in biologically aggressive HNSCC and suggest potential targets for therapy in CR cancers.