Aberrant activation of the Raf-MEK-ERK pathway has frequently been associated with various cancers, especially lung cancer. However, the key regulators of this pathway are largely unknown. Using functional proteomics screening, we found that KAP1 interacts with c-Raf. Knocking out KAP1 decreased c-Raf phosphorylation at serine 259 and increased its phosphorylation at serine 338, which activated MEK and ERK. We detected higher KAP1 expression in lung cancer tissues than in normal peri-tumoral tissues. KAP1 knockdown arrested A549 lung cancer cells in the G0/G1 phase of the cell cycle and attenuated cell growth, metastasis, the epithelial-mesenchymal transition, angiogenesis, stemness, and colony formation. Furthermore, knocking out KAP1 remarkably increased the susceptibility of A549 cells to the anti-cancer drug 5-Fluorouracil, which correlated with increasing ERK phosphorylation. In vivo xenograft experiments suggested that KAP1 deficiency significantly decreases the tumorigenicity of A549 cells. Taken together, our findings indicate that KAP1 acts as a key module in the c-Raf-interactome complex and regulates lung cancer development through the Raf-MEK-ERK pathway. Therefore, KAP1 may represent a potential diagnosis biomarker and new treatment target for lung cancer.