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Glioblastoma multiforme (GBM) is a type of brain tumor that is most aggressive, proliferates rapidly and intensive invasion is governed by cell migration and destruction of the extracellular matrix. In the present study, we evaluated the antiproliferative efficacy of the synthesized silver chloride nanoparticles (AgCl-NPs) from Asparagus racemosus root extract against human glioblastoma stem cells (GSCs) and Ehrlich ascites carcinoma (EAC) cells. Biosynthesis of A. racemosus-AgCl-NPs was confirmed by color change, UV–visible spectroscopy and characterized by transmitted electron microscope, energy dispersive spectroscopy, x-ray powder diffraction and Fourier-transform infrared spectroscopy. The A. racemosus-AgCl-NPs inhibited GSCs and EAC cells growth with the IC50 values of 4.8 and 4.69 µg/ml, respectively. A. racemosus-AgCl-NPs induced apoptosis in GSCs which was confirmed by annexin V/PI assay, various genes expression, and caspase-3 protein expression as detected by the immunofluorometric assay. The expression level of the TLR9, NFκB, TNFα, p21 and IKK genes were increased consequently with the decrease of PARP, EGFR, NOTCH2, mTOR and STAT3 genes in GSCs as examined by real-time PCR. The cell cycle arrest at G0/G1 phase was detected by flow cytometry. In addition, A. racemosus-AgCl-NPs caused significant inhibition of EAC cells growth, reduced tumor burden, increased the survival of EAC-bearing mice and restored the hematological parameters when compared with the control mice. The synthesized AgCl-NPs inhibited the proliferation of GSCs in vitro with the induction of apoptosis and inhibited the growth of EAC cells in vivo in mice by reducing the tumor burden and increasing the survival periods.