The high expression level of Neuropillin-1 (NRP1) is correlated with more advanced stages of metastasis and tumor progression in many epithelial carcinomas including breast cancer. However, the exact molecular mechanism by which it promotes breast cancer cell tumorigenicity is not well defined yet. NRP1 is a multifunctional protein that acts as a co-receptor for a number of growth factors including vascular endothelial growth factor VEGF-165 and transforming growth factor beta (TGFB1). In this study, we hypothesized that NRP1 can be a potential biomarker for breast cancer and might be involved in the Epithelial to Mesenchymal Transition (EMT) pathway. We investigated the functional role of NRP1 in three representative breast cancer cell lines in vitro. This was achieved by two main approaches; overexpressing and knocking down NRPI. NRP1 was overexpressed in the breast cancer cell lines MCF-7 and BT-474 known to express a moderate and small amount of the corresponding protein, respectively. The full length NRPI cDNA as well as the cDNA encoding the soluble form (S12NRPI) were cloned in PPTuner IRES2 plasmid. Moreover, we stably knocked down NRPI in MDA-MB-231 (expresses excessive amount of NRPI) cells using two different shRNA sequences targeting regions of this gene. Western blot results showed that NRP1 was significantly overexpressed in BT-474 compared to MCF-7 cells upon NRPI PPTuner (full length cDNA construct) transfection. In contrast, a significant reduction in NRP1 expression in MDA-MB-231 cells was observed upon transfection with shNRP1-06-1 plasmid. The proliferation alamarblue assay results showed unexpected trend of reduction in cellular proliferation in both transfected cells (MCF-7pPTuner and BT-474 PPTuner) whereas, a significant reduction in cellular proliferation of MDA-MB-231 cells was observed upon transfection with the same plasmid that cause down regulation of NRP1 expression in these cells. The expression level of NRP1 and some other related genes in the EMT and angiogenesis pathways (VEGF, TGFB1, ZEBI, PLGF, PDGF-A, PDGF-B, FGFRla and FGFRIB) were checked using RT-PCR. The results ated that mRNA gene expression of NRP1 and TGF1 showed a trend of decrease in expression between three cell lines in which MDA-MB-231 express high levels of the gene followed by the MCF7 and BT474 respectively. The expression of all the other checked genes except PDGFB was significantly higher in MDA-MB-231 cells compared to the other two non-metastatic cell lines. Our findings highlight the possible involvement of NRP1 in the EMT pathway since its expression level was proportionally increasing in the more tumorogenic cells and had similar pattern to those expressed levels of TGF-B and ZEB-1. Achieving complete silencing of its expression might inhibit breast cancer cell proliferation and therefore, it may serve as a useful prognostic biomarker for breast cancer diagnostics and a good target for an effective cancer therapy.