Breast cancer (BC) is the most common malignant tumor in women worldwide, and exploring new therapeutic targets is a great challenge for BC. SERTA domain containing 4 (SERTAD4) is a member of the SERTAD family, also known as Trip-Br or SEI proteins. The current biological function of SERTAD4 and its role in BC are unclear. The TNMplot, GEPIA2, HPA, TCGA, and Kaplan-Meier Plotter databases and clinical BC samples were used to analyze SERTAD4 expression-clinical correlations in BC. Single-cell sequencing data for BC were obtained from the GEO database to explore the expression, cellular localization, and biological function of SERTAD4 in BC. Additionally, the in vitro effects of siRNA knockdown of SERTAD4 on BC cells were investigated. The TNMplot and GEPIA2 databases indicate that SERTAD4 is upregulated in BC. The HPA database reveals that SERTAD4 protein is primarily localized in the cytoplasm. Analysis of TCGA data demonstrates a negative correlation between SERTAD4 expression and both estrogen receptor (ER) and progesterone receptor (PR) status. These database-derived patterns were consistently observed in our clinical cohort of BC specimens. The Kaplan-Meier Plotter database suggests that high SERTAD4 expression is associated with poor prognosis in BC. Single-cell analysis shows that SERTAD4 is predominantly expressed in epithelial cell subsets, with its expression level correlating with the degree of copy number variation (CNV). Functional enrichment analyses indicate that SERTAD4 can regulate the proliferation and adhesion of epithelial cells and is linked to the PI3K/AKT signaling pathway. In vitro knockdown of SERTAD4 inhibits the migration, invasion, and proliferation of BC cells, accompanied by downregulation of PI3K and AKT protein expression. Molecular docking and Co-immunoprecipitation (Co-IP) analyses revealed an interaction between SERTAD4 and PI3K. The upregulated expression of SERTAD4 in BC is associated with the prognosis of BC and is a potential prognostic factor and therapeutic target for BC.