Laryngeal carcinoma is a type of head and neck carcinoma with a high incidence and mortality. Chemotherapy treatments of human laryngeal carcinoma may fail due to the development of chemoresistance. Tissue inhibitor of metalloproteinase 3 (TIMP-3) has been shown to be implicated in a number of pathological processes typical for cancer. The present study aims to investigate the involvement of TIMP-3 in the chemoresistance of laryngeal carcinoma. We showed that TIMP-3 expression was significantly decreased in chemoresistant laryngeal carcinoma tissues compared with chemosensitivity tissues. Patients with low TIMP-3 expression exhibited poorer overall survival than those with high TIMP-3 expression. Moreover, cisplatin-resistant Hep-2 cells (Hep-2/R) were associated with the inhibition of mitochondrial membrane potential (MtMP) depolarization after cisplatin challenge. In addition, cisplatin resulted in a more pronounced mitochondrial cytochrome c release into the cytoplasm in Hep-2 cells than in their resistant variants. Overexpression of TIMP-3 by an adenovirus encoding TIMP-3 cDNA remarkably enhanced cisplatin-induced apoptosis, cytochrome c release, and caspase activation in Hep-2/R cells, thereby sensitizing cancer cells to cisplatin. On the other hand, downregulation of TIMP-3 markedly inhibited cisplatin-induced apoptosis in Hep-2 cells through attenuating mitochondria-dependent pathway activation. Taken together, these results demonstrate that decreased TIMP-3 expression may contribute to cisplatin resistance via inhibition of mitochondria-dependent apoptosis, indicating that forced TIMP-3 expression may be a useful strategy to improve the efficacy of cisplatin to treat laryngeal carcinoma.