Lung cancer is the leading cause of death worldwide and associated with dismal prognoses. As a major mitochondrial deacetylase, SIRT3 regulates the activity of enzymes to coordinate global shifts in cellular metabolism and has important implications for tumor growth. Its role as a tumor suppressor or an oncogene in lung cancer is unclear, especially in non-small cell lung carcinoma (NSCLC). To identify the mechanism of SIRT3-interacting proteins, we performed a yeast two-hybrid screen using a human lung cDNA library. One of the positive clones encoded the full-length cDNA of the nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) gene and the interaction between SIRT3 and NMNAT2 was identified. The interaction on growth, proliferation, apoptosis of NSCLC cell lines, and energy metabolism related to SIRT3 were investigated. Screening from the library resulted in NMNAT2 gene. We found that NMNAT2 interacts with SIRT3 both in vitro and in vivo; SIRT3 binds to NMNAT2 deacetylating it. Downregulation of SIRT3 inhibited acetylation of NMNAT2 and NAD+ synthesis activity of the enzyme. Low expression of SIRT3 significantly inhibited mitotic entry, growth and proliferation of NSCLC cell lines and promoted apoptosis, which was related to energy metabolism involving in the interaction between SIRT3 and NMNAT2. Taken together, our results strongly suggest that the binding of SIRT3 with NMNAT2 is a novel regulator of cell proliferation and apoptosis in NSCLC cell lines, implicating the interaction between SIRT3 and NMNAT2, energy metabolism associated with SIRT3.