Objective To investigate the expression and upstream/downstream mechanisms of ubiquitin fold modifier 1-specific peptidase domain protein (ZUP1) in breast cancer. Methods Gene information and clinicopathological data of breast cancer patients were retrieved using Gene Expression Omnibus (GEO), Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases. The relationships between the expression of ZUP1 and clinicopathological factors/survival status of breast cancer patients were analyzed by χ² test and Kaplan-Meier survival analysis, respectively. Bioinformatics methods were used for prediction of miRNAs and ubiquitin ligase that could potentially regulate ZUP1. Finally, the gene set enrichment analysis (GSEA) was performed. Results The expression of ZUP1 was higher in breast cancer tissues than in normal control tissues, and was related to T stage, PAM50 classification, statuses of estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2, and histological type of breast cancer (all P<0.01). The overall survival time of patients with high expression of ZUP1 was significantly lower than that of patients with low expression of ZUP1 (P=0.031). Bioinformatics predicted that the top 10 miRNAs targeting ZUP1 with the highest differential expression were miRNA-10b-3p, miRNA-499a-5p, miRNA-181b-2-3p, miRNA-181b-3p, miRNA-4420, miRNA-548aw, miRNA-5680, miRNA-570-3p, miRNA-7156-5p and miRNA-8087. E3 ubiquitin ligase including MARCH1, MARCH8, Mdm2, synoviolin and MIB1 may regulate the expression of ZUP1. The GSEA results indicated that ZUP1 was mainly involved in basic transcription factor, ubiquitin mediated proteolysis, oocyte meiosis, RNA degradation and Aurora B pathway. Conclusion The expression of ZUP1 is up-regulated in breast cancer, and is related to prognosis. The upstream and downstream mechanisms of ZUP1 in the development and progression of breast cancer are related to a variety of miRNAs and multiple signaling pathways.