Glioblastoma (GBM) is the most malignant and aggressive glioma, which has a very poor prognosis. Temozolomide (TMZ) is still a first‐line treatment, but resistance is inevitable even in MGMT‐deficient glioblastoma cells. The aims of this study were to comprehend the effect of TMZ on nucleus and the underlying mechanism of acquired TMZ resistance in MGMT‐deficient GBM. We show the changes of nuclear proteome in the MGMT‐deficient GBM U87 cells treated with TMZ for 1 week. Label‐free–based quantitative proteomics were used to investigate nuclear protein abundance change. Subsequently, gene ontology function annotation, KEGG pathway analysis, protein‐protein interaction (PPI) network construction analysis of DAPs, and immunofluorescence were applied to validate the quality of proteomics. In total, 457 (455 gene products) significant DAPs were identified, of which 327 were up‐regulated and 128 were down‐regulated. Bioinformatics analysis uncovered RAD50, MRE11, UBR5, MSH2, MSH6, DDB1, DDB2, RPA1, RBX1, CUL4A, and CUL4B mainly enriched in DNA damage repair related pathway and constituted a protein‐protein interaction network. Ribosomal proteins were down‐regulated. Cells were in a stress‐responsive state, while the entire metabolic level was lowered.Significance of the studyIn U87 cell treated with TMZ for 1 week, which resulted in DNA damage, we found various proteins dysregulated in the nucleus. Some proteins related to the DNA damage repair pathway were up‐regulated, and there was a strong interaction. We believe this is the potential clues of chemotherapy resistance in tumour cells. These proteins can be used as indicators of tumour resistance screening in the future.