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Recently, the preparation of silver nanoparticles (AgNPs) by green technique has improved due to their fundamental applications in medicine. In this study, methadone syrup (ME) was used for the preparation of AgNPs as a reducing and stabilizing agent with the aim of in vitro cytotoxicity effect against the human breast cancer cells. The characteristics of prepared particles are investigated by transmission electron microscopy (TEM), environmental scanning electron microscopy (ESEM), energy-dispersive X-ray spectroscopy (EDS), dynamic light scattering (DLS), UV-visible spectroscopy (UV-Vis), Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction studies (XRD). The AgNPs (about 18 nm) were synthesized in a spherical shape and uniform distribution. The mechanism of ME through the synthesis has been proposed based on FT-IR analysis and density functional theory (DFT). To investigate the cytotoxicity of prepared AgNPs by ME, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used in the range of 0-100 μg/mL. As a function of its dosage, the green synthetic AgNPs showed antiproliferation activity against MDA-MB-468 cells respect to ME. The results demonstrated the feasibility of producing AgNPs in a simple, rapid, and green manner using ME, which has an important function in inhibiting the growth of breast cancer cells.