Targeted inhibition of mutated kinases using selective MAP kinase inhibitors in malignant melanoma often results in temporary improvement of clinical symptoms followed by rapid development of resistance. To gain insights in molecular processes that govern resistance, we performed SILAC-based quantitative proteomics profiling of vemurafenib-resistant and -sensitive melanoma cells. Among downregulated proteins in vemurafenib-resistant cell lines we detected multiple proteins involved in cytoskeletal organization and signaling, including the intermediate filament nestin, which was one of the most downregulated proteins. Previous studies showed that nestin is expressed in various types of solid tumors and its abundance correlates with malignant phenotype of transformed cells. However, the role of nestin in cancer cells regarding acquired resistance is still poorly understood. We performed CRISPR/Cas9 knockout of the nestin gene (NES) in vemurafenib-sensitive cells and showed that loss of nestin leads to increased cellular proliferation and colony formation upon treatment with BRAFV600E and MEK inhibitors. Moreover, nestin depletion led to increased invasiveness and metalloproteinase activity like the phenotype of melanoma cells with acquired resistance to the BRAF inhibitor. Finally, phosphoproteome analysis revealed that nestin depletion influenced signaling through integrin and PI3K/AKT/mTOR pathways and led to increased focal adhesion kinase abundance and phosphorylation. Taken together, our results reveal that nestin is associated with acquired vemurafenib resistance in melanoma cells.