Breast cancer is the most commonly diagnosed type of cancer and one of the leading causes of cancer-associated mortality in women. In addition, the underlying molecular mechanisms of the occurrence and development of breast cancer requires further investigation. In the present study, bioinformatics analysis was performed to identify differentially expressed genes (DEGs) between breast cancer and normal breast tissues to investigate the underlying molecular mechanisms. In addition, reverse transcription-quantitative PCR and immunohistochemistry (IHC) were performed to investigate the protein and mRNA expression levels of a specific DEG, discs large-associated protein 5 (DLGAP5). A Cell Counting Kit-8 assay and flow cytometry analysis were used to assess the effects of DLGAP5 on cell proliferation. In total, 85 DEGs were identified in the three Gene Expression Omnibus datasets, including 40 upregulated and 45 downregulated genes. In addition, 30 hub genes were identified following the construction of a protein-protein interaction network, and 28 of the 30 hub genes were established to be indicators of breast cancer prognosis. DLGAP5 was highly expressed in breast cancer specimens, and its expression levels were correlated with clinical stage and lymph node status. In addition, downregulation of DLGAP5 repressed the proliferation of breast cancer MDA-MB-231 cells and induced cell cycle arrest. Additionally, DLGAP5 was identified to be localized in the mitochondria, and the presence of a conserved microtubule-associated proteins 1A/1B light chain 3B-interacting region motif suggested that DLGAP5 may serve a role in mitophagy. The present results demonstrated an association between DLGAP5 expression levels and the clinicopathological characteristics of patients with breast cancer using IHC. In conclusion, DLGAP5 may be a promising target in the diagnosis and treatment of breast cancer.