Mitoxantrone is one kind of chemical therapy medicine for cancer but certain kinds of cancer cells are chemical-resistant to it. In this research, we analyzed the quantitative proteomic difference between tumors in vivo xenograft by mitoxantrone-resistant (M group) and wild NCI-H460 cells (N group). Protein expression profiling in combination with pathway analysis was deployed to investigate molecular events associated with the tumors using a label-free quantitative proteomic approach. A total of 173 proteins were significantly differentially expressed in mitoxantrone-resistant tumors. Bioinformatics analysis using the cytoscape platform indicated that biological processes, including actin-mediated cell contraction, muscle system process, muscle filament sliding, and muscle contraction, are involved in mitoxantrone-resistance. As KEGG pathway enrichment analysis has shown, systemic lupus erythematosus, alcoholism, viral carcinogenesis, and tight junction are strongly regulated with chemical-resistance. By protein-protein interaction analysis, three protein clusters were found using k-means clustering algorithm. Dysregulation results can be verified by Western blotting. Further studies into the molecular functions of dysregulated proteins will help to provide new perspectives regarding chemoresistance for non-small cell lung cancers.