In this study, expression of the SPC25 gene was characterized in breast cancer (BC), and its effects on BC development and progression, functions in BC cells, and potential underlying mechanisms were examined. Data from TCGAportal and FIREBROWSE indicated that SPC25 was upregulated in BC tissues compared to normal tissues, and CANCERTOOL indicated that higher SPC25 mRNA levels were associated with increased probability of recurrence and poorer survival in BC patients. BC patients with higher SPC25 expression displayed shorter distant metastasis-free survival, relapse-free survival, and overall survival. Colony formation and CCK-8 experiments confirmed that SPC25 promoted proliferation of BC cells. Single-cell analysis indicated that SPC25 is associated with cell cycle regulation, DNA damage and repair, and BC cell proliferation. SPC25 knockdown suppressed proliferation of BC cells. MiRNAs, circRNAs, RNA-binding proteins, transcription factors, and immune factors that might interact with SPC25 mRNA to promote BC were also identified. These findings suggest that SPC25 levels are higher in more malignant BC subtypes and are associated with poor prognosis in BC patients. In addition, DNA methyltransferase inhibitor and transcription factors inhibitor treatments targeting SPC25 might improve survival in BC patients.