Daidzein (DZ), a water-insoluble isoflavone, has many beneficial effects (anti-inflammatory, antioxidant, and anticancer effects, etc.) on human health. DZ has a very low oral bioavailability related to its physicochemical properties (low solubility, intense metabolism of DZ in the intestine and liver). This study aimed to prepare and in vitro characterize the nanosuspension formulations of DZ to improve the poor solubility and efficacy of DZ. DZ nanosuspension formulations were prepared with media milling technique using zirconium oxide beads as milling media. Pluronic F127 and polyvinylpyrrolidone (PVP) K30 (formulation A; F-A) and sodium dodecyl sulfate (SDS) (SDS + pluronic F127 + PVP K30; formulation B; F-B) were used as stabilizers. The nanosuspension formulations were evaluated for morphological properties, particle sizes, zeta potential, DZ content, saturation solubility, dissolution, and their cytotoxic effects on RG2 glioblastoma tumor cells. F-A and F-B formulations were nanosized (in the range of about 181-235 nm) and had negative zeta potential values before and after lyophilization. The DZ content of F-A and F-B formulations were found to be 93.68±0.78% and 89.75±0.49%, respectively. Fourier transform infrared spectroscopy analysis showed that there was no significant interaction between DZ and the excipients. Differential scanning calorimetry and X-ray diffraction analyses confirmed no change in the crystal structure of DZ in F-A and F-B formulations. In this study, the nanosuspension formulations were successfully prepared and characterized in vitro. Nanosuspension formulations increased the saturation solubility, dissolution rate, and cytotoxic effect of DZ.