AbstractBreast cancer is the most common type of cancer worldwide. However, the well-known molecular biomarkers are not enough to meet the needs of precision medicine. In search for novel targets in this regard, we reported ITSN1 (intersectin1) as one of the candidates through mRNA microarray analysis. In the present study, we reported that endocytic protein ITSN1-S exists not only in the cytoplasm but also in nuclei of breast cancer cells. ITSN1-S′ functional nuclear localization signal is within its residues 306–312. Its nuclear export signal (NES) resides within its SH3 domains. We also found, the interaction between the CC domain of nuclear ITSN1-S and the NT domain of nuclear DNA helicase II (NDH II) directly suppressed the DNA replication and nascent DNA synthesis by inhibiting the R-loops resolution in breast cancer cells. Furthermore, the interaction between the EH domains of cytoplasmic ITSN1-S and PI3KC2α inhibit cell migration and invasion by inactivating the PI3KC2α-AKT pathway. Our results were confirmed in both ITSN1 gene knockout cells and in vivo assays. Finally, our clinical data showed a potential application of the combined consideration of the cytoplasmic and nuclear ITSN1-S as an independent prognosis factor. In conclusion, our study revealed ITSN1-S′ novel positioning in the nuclei of breast cancer cells, its function in suppressing DNA replication, and its potential application in improved breast cancer prognosis.