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A green method for synthesizing gold nanoparticles is proposed using hydroethanolic extract of Vitex mollis fruit (Vm extract) as a reducer and stabilizer. The formation of gold nanoparticles synthesized with Vm extract (AuVmNPs) was monitored by measuring the ultraviolet-visible spectra. The morphology and crystalline phase were determined using scanning electron microscopy, X-ray diffraction, and high-resolution transmission electron microscopy. Synthesized nanoparticles were generally spherical, and the size distribution obtained by transmission electron microscopy shows two populations with mean sizes of 12.5 and 22.5 nm. Cell viability assay using MTT and cellular apoptosis studies using annexin V on human umbilical vein endothelial cells (HUVECs) and the human mammary epithelial cell line (MCF10A) indicate that AuVmNPs have low toxicity. Cell migration tests indicate that AuVmNPs significantly inhibit HUVEC cell migration in a dose-dependent manner. The evaluation of the localization of AuVmNPs in HUVECs using confocal laser scanning microscopy indicates that nanoparticles penetrate cells and are found in the cytosol without preferential distribution and without entering the nucleus. The inhibitory effect on cellular migration and low toxicity suggest AuVmNPs as appropriate candidates in future studies of antiangiogenic activity., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)