Homeobox (HOX) transcript antisense RNA (HOTAIR), a long nuclear-retained noncoding RNA (lncRNA), is overexpressed in a variety of human cancers. Increasing evidence shows that HOTAIR plays a vital role in cancer initiation and progression by affecting cell cycle progress, apoptosis and invasion. However, whether HOTAIR serves as a target of therapeutic potential and the underlying mechanism in head and neck squamous cell carcinoma (HNSCC) is still unclear. Thus, we employed a HOTAIR specific siRNA to deplete its expression in two human HNSCC cell lines, Tca8113 and Tscca. The flow cytometry (FCM) analysis showed that HOTAIR depletion induced tumor cell apoptosis in vitro. JC-1 probe examination showed that the mitochondrial membrane potential was changed significantly by HOTAIR blockage. Mitochondrial calcium uptake 1(MICU1) dependent cell death was induced by HOTAIR depletion. Protein expression analysis indicated that mitochondrial related cell death pathway (Bcl-2, BAX, Caspase-3, Cleaved Caspase-3, Cytochrome c) involved in HOTAIR dependent apoptosis process. Moreover, a Tscca derived xenograft tumor model was employed to further validate that injection of HOTAIR siRNA inhibited tumor growth. In summary, we suggested that HOTAIR inhibition could be developed as a new therapeutic in HNSCC treatments.