Transcription factor Krüppel-like factors 5 (KLF5) is overexpressed in a wide range of tumor tissues and acts as a prognostic factor in cancer. However, the role of KLF5 in non-small cell lung cancer is not clear. Hypoxia plays a vital part in the development of cancer via hypoxia-inducible factor 1 (HIF-1). Our study showed that hypoxia (1% O2) increased cell viability, clonality and proliferation and inhibited cell apoptosis in A549 cells. The expression of HIF-1α and KLF5 was increased time-dependently in hypoxia. Using small interfering RNA (siRNA) targeting KLF5 or HIF-1α, we demonstrated that KLF5 or HIF-1α knockdown inhibited hypoxia-induced cell survival and promoted cell apoptosis by actively downregulating cyclin B1, survivin and upregulating caspase-3. Given the similar effect of KLF5 and HIF-1α on cell survival, an attempt was made to investigate the putative interaction of them in hypoxia. KLF5 was revealed to co-immunoprecipitate with HIF-1α and hypoxia increased the amount of KLF5 and HIF-1α complex. Moreover, silencing of KLF5 decreased HIF-1α expression while KLF5 was not affected by HIF-1α inhibition in hypoxia, confirming the effect of KLF5 on upregulation of HIF-1α. In conclusion, this study identified hypoxia as a tumor promoter by triggering KLF5 → HIF-1α → cyclin B1/survivin/caspase-3 in lung cancer cells.