The purposes of this study were to investigate the effects of phosphatidylethanolamine-binding protein 4 (PEBP4) on the cell growth, proliferation, apoptosis, and invasion of non-small cell lung cancer (NSCLC) cells and to provide evidence for future treatment options for NSCLC. Western blot assays were performed to examine PEBP4 protein expression levels in NSCLC cell lines (HCC827, A549, NCI-H661, NCI-H292, and 95-D) and a normal human bronchial epithelial (HBE) cell line. A PEBP4 shRNA expression vector was constructed and transfected into HCC827 cells. Subsequently, the effects of PEBP4 on the cell viability, cell cycle distribution, apoptosis levels, and invasion properties of HCC827 cells were analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, flow cytometry analyses, and transwell invasion assays. In addition, the effects of PEBP4 on the expression of proteins including cyclin D1, p53, Bcl-2, MMP-2, and MMP-9 were investigated. PEBP4 was highly expressed in lung cancer cells (HCC827, A549, NCI-H661, NCI-H292, and 95-D), but its expression was low in HBE cells. Cell viability, cell proliferation, and invasion of HCC827 cells in the PEBP4 knockdown group were significantly lower than that in the negative control and blank control groups (p < 0.05), and there were no significant differences between the negative and blank control groups in terms of cell viability, cell proliferation, apoptosis, and invasion. In HCC827 cells, the expression levels of cyclin D1, Bcl-2, MMP-2, and MMP-9 in the PEBP4 knockdown group were significantly lower (p < 0.05), and the expression of p53 protein was significantly higher than that in the negative and blank control groups (p < 0.05). There were no significant differences between the negative and blank control groups in the expression levels of cyclin D1, p53, Bcl-2, MMP-2, and MMP-9. In conclusion, PEBP4 enhanced HCC827 cell proliferation and invasion ability and inhibited apoptosis. Decreased PEBP4 expression may play a role in the reduced invasion ability and increased apoptosis of the human NSCLC cell line HCC827.