OBJECTIVE: Tumor protein 53 (TP53) is the commonly mutated gene in non-small cell lung cancer (NSCLC) that is associated with poor prognosis, and anlotinib exerts inhibitory effects on TP53-mutated NSCLC. The aim of this study was to investigate the inhibitory effect of anlotinib on TP53-mutated NSCLC and its possible mechanism. METHODS: The growth ability of TP53-mutated NSCLC cells were tested by Cell counting kit-8 assay. Proteins in TP53-mutated NSCLC cells treated with anlotinib were analyzed using label-free liquid chromatography-mass spectrometry. Differentially represented proteins were analyzed by KEGG, GO, and PPIs. TP53 pathway related proteins were verified using western blotting. RESULTS: The cell viability was significantly reduced in TP53-mutated NSCLC cell as opposed to TP53 wild cell by anlotinib treatment. 126 differentially represented proteins (37 upregulated and 89 downregulated) were found between the anlotinib and control groups in TP53-mutated NSCLC cell. Bioinformatics analyses revealed that the differentially represented proteins were primarily involved in catalytic activity, cellular processes, and metabolite interconversion. PANTHER Classification System found that anlotinib mainly impacted the p53 signaling pathway, De novo purine biosynthesis and Integrin signaling. KEGG enrichment and PPI networks of the differentially represented proteins revealed cyclin-dependent kinase 1 (CDK1) and mitogen-activated protein kinase kinase 3 (MAP2K3) as the core protein, which are related to the p53 signaling pathway. Western blotting also revealed that anlotinib significantly suppressed the expression of CDK1 and MAP2K3 in TP53-mutated NSCLC cells, that indicated the possible mechanism may involve the MAP2K3/p53/CDK1 pathway. CONCLUSIONS: Our findings showed that anlotinib selectively inhibited the growth of TP53-mutated NSCLC cells and downregulated the expression levels of CDK1 and MAP2K3. The MAP2K3/p53/CDK1 pathway may be the molecular mechanism underlying anlotinib's efficacy in TP53-mutated NSCLC. STATEMENT OF SIGNIFICANCE: Tumor protein 53 (TP53) is the commonly mutated gene in non-small cell lung cancer (NSCLC) that is associated with poor prognosis, and anlotinib exerts inhibitory effects on TP53-mutated NSCLC. However, the action mechanism of anlotinib in the treatment of TP53-mutated NSCLC remains unclear. In this study, we used label-free quantitative proteomics to reveal the molecular mechanism of anlotinib inhibition in TP53-mutated NSCLC. We found that anlotinib significantly inhibited the growth of TP53-mutated NSCLC cells and downregulated the expression levels of CDK1 and MAP2K3. The MAP2K3/p53/CDK1 pathway may be the molecular mechanism underlying anlotinib's efficacy in TP53-mutated NSCLC. Our study promotes the use of anti-angiogenic drugs in TP53-mutated NSCLC. It provides new ideas for the treatment of TP53-mutated NSCLC.