Nasopharyngeal carcinoma (NPC) is a major malignant tumor of the head and neck region in southern China. The understanding of its underlying etiology is essential for the development of novel effective therapies. We report for the first time that microRNA-940 (miR-940) significantly suppresses the proliferation of a variety of cancer cell lines, arrests cells cycle, induces caspase-3/7-dependent apoptosis and inhibits the formation of NPC xenograft tumors in mice. We further show that miR-940 directly binds to the 3'-untranslated regions of Nestin mRNA and promotes its degradation. Likewise, depletion of Nestin inhibits tumor cell proliferation, arrest cells at G2/M, induces apoptosis and suppresses xenograft tumor formation in vivo. These functions of miR-940 can be reversed by ectopic expression of Nestin, suggesting that miR-940 regulates cell proliferation and survival through Nestin. Notably, we observed reduced miR-940 and increased Nestin levels in NPC patient samples. Protein microarray revealed that knockdown of Nestin in 5-8F NPC cells alters the phosphorylation of proteins involved in the DNA damage response, suggesting a mechanism for the miR-940/Nestin axis. Consistently, depletion of Nestin induced spontaneous DNA damage accumulation, delayed the DNA damage repair process and increased the sensitivity to irradiation and the chemotherapeutic agent doxorubicin. Collectively, our findings indicate that Nestin, which is downregulated by miR-940, can promote tumorigenesis in NPC cells through involvement in the DNA damage response. The levels of microRNA-940 and Nestin may serve as indicators of cancer status and prognosis.