Background <p>Anlotinib is an effective treatment for advanced non-small cell lung cancer (NSCLC), but resistance to it often develops during therapy. RAF1, a serine/threonine kinase involved in cancer progression, has limited research in NSCLC, particularly regarding anlotinib resistance.</p> Methods <p>Analysis of RAF1 expression in NSCLC and its relationship with targeted therapy resistance and apoptosis through bioinformatics methods. Immunohistochemistry (IHC) was employed to evaluate the relationship between RAF1 expression and anlotinib resistance in NSCLC tissues. Anlotinib-resistant PC9 (PC9/AR) cells were constructed in vitro, and cell viability and apoptosis were assessed using the cell counting kit-8 (CCK-8) assay and flow cytometry. Quantitative real-time PCR (qRT-PCR) was carried out to evaluate RAF1 gene expression levels, and western blot (WB) analysis was conducted to determine the expression of RAF1, Bcl-2-associated X protein (Bax) and B-cell lymphoma 2 (Bcl-2).</p> Results <p>Bioinformatics analysis showed that RAF1 was lowly expressed in lung cancer tissues in TCGA and GEPIA databases. Further pathway analysis indicated that RAF1 expression was positively correlated with targeted therapy resistance and negatively correlated with the expression of the anti-apoptotic protein Bcl-2. Immunohistochemical analysis showed that high RAF1 expression in NSCLC tissues was related to anlotinib resistance (P &lt; 0.05). In vitro experiments demonstrated that RAF1 contributed to anlotinib resistance in NSCLC cells. Overexpression of RAF1 increased cell viability and decreased apoptosis in PC9 and PC9/AR cells, while knockdown of RAF1 had the opposite effects.</p> Conclusion <p>RAF1 mediates anlotinib resistance in NSCLC cells by regulating apoptosis and may serve as a predictive marker for anlotinib resistance in advanced lung cancer patients.</p>