Objective Nanomaterials with superior properties such as high surface area over volume ratio are widely used in dentistry and medicine. This in vitro study was performed to synthesize and characterize nano bioactive glass (nBG) and to evaluate the effect of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) containing nBG (CPP-ACP@nBG) on enamel remineralization by its application to pH-cycled, synthetically demineralized enamel surfaces. Materials and methods nBG particles were prepared by sol-gel method. X-ray diffraction pattern (XRD), Fourier-transform infrared spectroscopy (FTIR) and transmittance electron microscopy (TEM) were used for nBG characterization. Synthetic CPP-ACP paste was prepared and nBG particles were added to it. To evaluate the degree of remineralization, 32 healthy human premolars were selected. The samples were randomly divided into 4 groups as: Group 1: Commercial CPP-ACFP (MI paste plus), Group 2: Synthetic casein phosphopeptide-amorphous calcium phosphate containing fluoride (CPP-ACP@F), Group 3: Synthetic CPP/ACP containing nBG (CPP-ACP@nBG), and Group 4: Control (received no treatment). The pastes were then applied on the tooth surfaces for 28 days. The Vickers microhardness of enamel surfaces was evaluated, and enamel surface morphology was assessed using scanning electron microscopy (SEM). Results X-Ray diffraction pattern (XRD) of the synthesized nBG show its crystalline nature with the Larnite crystalline mode. Transmittance electron microscope (TEM) microimage of the synthesized nBG shows its formation as less that 100 nm spherical nanoparticle with partial agglomeration. Fourier transform infrared spectroscopy (FTIR) confirm the success formation of nBG with high purity. The results of this study showed that microhardness of the experimental groups was significantly higher than the control group (p ≥ 0.05). SEM images showed a layer of hydroxyapatite in the CPP-ACP@nBG, synthetic and commercial CPP-ACP@F remineralized groups. Conclusion The results of this study demonstrated that CPP-ACP@F and CPP-ACP@nBG remineralize the surface of the demineralized enamel. Microhardness of the remineralized enamel in the CPP-ACP@nBG group was higher than synthetic and commercial CPP-ACP@F groups.