Background: Metformin, a first-line antidiabetic drug, has recently been reported with anticancer activities in various cancers; however, the underlying mechanisms remain elusive. The aim of the present study was to investigate the role of cellular FADD-like IL-1 beta-converting enzyme (FLICE)-inhibitory protein large (c-FLIPL) in metformin-induced anticancer activity in non-small cell lung cancer (NSCLC) in vitro. Materials and methods: Cell viability was measured by MTT assay. Quantitative real-time PCR was carried out to detect the level of mRNA of related genes. The expression of related proteins was detected by Western blot. siRNA was used to silence the expression of targeted proteins. Results: Metformin significantly suppressed proliferation of both A549 and H460 cells in a dose-dependent manner. Mechanistic studies suggested that metformin killed NSCLC cells by inducing apoptotic cell death. Moreover, metformin greatly inhibited c-FLIPL expression and then promoted its degradation. Furthermore, metformin significantly activated Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) and its downstream glycogen synthase kinase 3beta (GSK-3 beta), block the expression of AMPK, and GSK-3 beta with siRNA partially reversed metformin-induced cytotoxicity and restored the expression of c-FLIPL in lung cancer cells. Metformin also suppressed the activity of AMPK downstream protein kinase A (PKA), PKA activators, both 8-Br-cAMP and forskolin, greatly increased c-FLIPL expression in NSCLC cells. Conclusion: This study provided evidence that metformin killed NSCLC cells through AMPK/PKA/GSK-3 beta axis-mediated c-FLIPL degradation.