Background: Prostate cancer is a major cause of disease and mortality among men. GNT is an isoflavone found naturally in legumes. Isoflavones, a subset of phytoestrogens, are structurally similar to mammalian estrogens. This study aimed to evaluate the anticancer and cytotoxic effects of GNT on PC3 cell line under 3D culture medium. Methods: The 3D culture was created by encapsulating the PC3 cells in alginate hydrogel. MTT assay, neutral red uptake, comet assay, and cytochrome C assay were used to study the anticancer and cytotoxic effects of GNT at 120, 240, and 480 μM concentrations. Also, NO, catalase, and GSH levels were determined to evaluate the effect of GNT on the cellular stress. The culture medium was used as the negative control. Results: GNT reduced the production of cellular NO and increased the production of catalase and glutathione, confirming the results of the NO test. Evaluation of the toxicity effect of GNT at the concentrations of 120, 240, and 480 μM using comet assay showed that this chemical agent induces apoptosis in PC3 cells in a dose-dependent manner. As the level of cytochrome C in PC3 cells treated with different concentrations of GNT was not significantly different from that of the control, GNT could induce apoptosis in PC3 cells through the non-mitochondrial pathway. Conclusion:The findings of this study disclose that the anticancer effect of GNT on PC3 cells under 3D culture conditions could increase the effectiveness of treatment. Also, the cell survival rate is dependent on GNT concentration.