Aim The aim of this study was to determine whether gold nanoparticles conjugated cytotoxic protein NKCT1 (GNP-NKCT1) acted through the estrogen receptor mediated pathway in MCF-7 cells and to establish the MAPK and PI3k/Akt signal transduction pathway. Methods Apoptosis was done by flow cytometry. BrdU incorporation and nuclear proliferating antigen was measured by flow cytometry. Wound healing assay along with matrigel chamber invasion and migration was done. Expression of MMP9 was checked by flow cytometry and also by gelatin zymography. To analyze the regulation of signaling protein, western blot was done. MTT assay was done to evaluate the ligand receptor pathway using the estrogen receptor negative cell line (MDA-MB-231) for inhibitor effects. Results Treatment of GNP-NKCT1 (3.9 μg/ml) exhibited 38.04% early apoptosis and 4.29% late apoptotic cell. GNP-NKCT1 significantly inhibited both cell migration and invasion with suppressed expression of MMP9. In addition, treatment of cultured human breast cancer MCF7 cells with GNP-NKCT1 reversely suppressed the incorporation of BrdU, with reduced expression of Ki-67. The western blot analysis showed that GNP-NKCT1 arrested cell cycle progression through upregulation of the kinase inhibitor protein p21 and inactivation of G1-cylin dependent kinase (CDK4). GNP-NKCT1 suppressed nuclear translocation of nuclear factor kappa B (NF-κB) and also abrogated the phosphorylation of p38 mitogen activated protein kinase (MAPK), phosphatidylinositide-3-kinase (PI3k), Akt and extracellular regulated kinase (ERK1/2). MTT assay indicated that GNP-NKCT1 reduced proliferation in the estrogen receptor induced ER negative breast cancer cell line (MDA-MB-231). Addition of, ER inhibitor (tamoxifen) and PI3K inhibitor (wortmannin) to cells resulted in reduced expression of Ki-67 and MMP-9. Conclusion The data suggested that GNP-NKCT1 induced MCF7 cell inhibition may occur through estrogen receptor pathway via inactivation of CDK4 and inactivation of PI3K/Akt, ERK1/2 and p38 MAPK signaling pathway with inhibitory effects on NF-κB, reducing the activity of MMP9. This result provides a new mechanism to explain the role of gold nanoparticles conjugated NKCT1 as a potent anti-metastatic agent in MCF7 cells. © 2017 Elsevier B.V.