BACKGROUND The actin filament-associated protein (AFAP) family consists of 3 novel adaptor proteins: AFAP1, AFAP1L1, and AFAP1L2/XB130. Although evidence shows that AFAP1 and AFAP1L2 play an oncogenic role, the effect of AFAP1L1 on tumor cell behavior has not been fully elucidated, and it remains unknown whether AFAP1L1 could be a prognostic marker and/or therapeutic target of lung cancer. MATERIAL AND METHODS Human A549 non-small cell lung cancer (NSCLC) cells were used in this study. AFAP1L1 gene was knocked down by AFAP1L1 short hairpin RNA (shRNA) transfection. Cell proliferation was analyzed using Celigo image cytometry and MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay, cell cycle progression was assessed with flow cytometry, and cell apoptosis was determined by flow cytometry after annexin-n staining. The PathScan intracellular signaling array was used to investigate cancer-related signaling proteins influenced by knocking down AFAP1L1 in A549. RESULTS AFAP1L1 gene expression was successfully inhibited by the AFAP1L1-shRNA transfection. Cell proliferation was inhibited and cell proportions in G1 and G2/M phases were increased, and cell apoptosis was increased in the AFAP1L1-shRNA transfected cells as compared with negative control shRNA transfected cells. Using the PathScan intracellular signaling array, we found that downregulation of AFAP1L1 significantly activated P38 and caspase 3, and inhibited PRAS40 activation. CONCLUSIONS Our data show that AFAP1L1 promotes cell proliferation, accelerates cell cycle progression, and prevents cell apoptosis in lung cancer cells. Therefore, AFAP1L1 might play an oncogenic role in NSCLC.