Hypoxia induced drug resistance is a major obstacle in the development of effective cancer therapy. Hypoxia is also associated with human non-small cell lung cancer (NSCLC), which is highly resistant to chemotherapy. However, the exact mechanisms driving hypoxia induced chemoresistance of NSCLC remain elusive. We first found that NSCLC cell lines showed hypoxia-induced chemoresistance from human several solid tumors treated with Cisplatin. Especially, IC50 values to NSCLC cell lines, H1299 and A549 are significantly more increased under hypoxic condition than these of normoxic condition. To evaluate why IC50 values are significantly increased in NSCLC cell lines, we measured the expression levels of SIRT1 and pAMPK alpha after normoxic and hypoxic condition 48 h with 21 solid tumors by using Western blot analysis. Surprisingly, the levels of SIRT1 and pAMPK alpha in NSCLC cell lines were highly underestimated in hypoxic condition. Cell viabilities, colony numbers, and IC50 values were diminished by SIRT1 overexpression and SIRT1 activator (SRT1720), which was recovered by AMPK alpha knock-down and AMPK alpha inhibitor (Compound C), and also these were increased by SIRT1 knock-down, SIRT1 mutant (H363Y) expression, and SIRT1 inhibitor (EX527), which was re-decreased by AMPK alpha expression and AMPK alpha activator (A769662). These results suggest that hypoxia-induced chemoresistance is correlation with SIRT1-AMPK alpha axis. To elucidate how SIRT1 can overcome hypoxia-induced chemoresistance, we measured mitochondria activity by using mitochondria morphology change, MitoTracker, and ATP/ADP ratio. SIRT1 expression increased the Area, number, perimeter, ferret’s meter, ATP/ADP ration of mitochondria, and promoted cytochrome C release from mitochondria, which was diminished by siAMPK alpha. Moreover, our new combination therapy SIRT1 activator and Cisplatin can overcome resistance to hypoxia-induced chemoresistance through activation of mitochondria activity. Actually, these regime synergistically decreased tumor volume and weight in H1299 xenograft model and significantly increased survival rate in H1299 lung orthotopic model in compared with either each single or non-treatment. Our results showed that SIRT1-AMPK alpha axis by chronic hypoxia makes chemoresistance via the inactivation of mitochondria. However, ectopically increased SIRT1 expression or activity in hypoxia can overcome hypoxia-induced chemoresistance via activated mitochondria. It suggests that SIRT1 is an efficient target for reducing chemoresistance in NSCLC. Key words: SIRT1, AMPK alpha, hypoxia, chemoresistance, mitochondria activity, Cisplatin