γ-radiation is widely utilized in medical and research applications, particularly in oncology, where it plays a crucial role in radiation therapy for eliminating malignant tumor cells [1]. However, its non-selective nature means that healthy cells can also suffer damage, leading to adverse side effects [2]. The harmful impact of γ-rays occurs through direct interactions with cellular macromolecules, such as DNA, causing strand breaks, or indirectly, by ionizing water molecules and generating highly reactive oxygen species (ROS) [3]. An imbalance between ROS production and the body's antioxidant defense results in oxidative stress, which is a key factor in radiation-induced cellular damage [4]. Moreover, γ-irradiation disrupts liver enzyme function, altering serum alkaline phosphatase (ALK-P), GPT, and GOT levels, as well as affecting total cholesterol and albumin concentrations [5,6]. To counteract the effects of radiation exposure, radioprotective agents have been developed to minimize oxidative stress, suppress free radical formation, and activate cellular defense mechanisms, including antioxidants [7]. These protective compounds not only reduce bystander effects but also enhance DNA repair pathways and inhibit apoptotic signalling, thus reducing radiation-induced injuries [8]. The search for an effective radioprotective compound dates back to 1948 when CYS was first identified for its ability to protect cells from radiation damage, drawing significant interest from the U.S. Army [9]. CYS, a sulfur-containing amino acid, has emerged as an important biochemical radioprotectant due to its ability to neutralize hydroxyl radicals and donate protons via its -NH2, -SH, and -COOH functional groups [10]. Additionally, CYS serves as a key precursor for GSH, one of the body's most critical antioxidants, which detoxifies ROS generated by γ-radiation [11]. By maintaining redox homeostasis, CYS plays a vital role in protecting biomolecules such as DNA, proteins, and lipids from oxidative damage. Furthermore, CYS directly scavenges free radicals and prevents oxidative lesions, reinforcing its potential as a radioprotective agent [12,13]. In addition to biochemical protectants, nanomaterials have shown significant potential in radiation shielding applications. ZFN belong to the spinel ferrite family, composed of zinc and iron oxides. These nanoparticles exhibit high atomic density, magnetic properties, and strong γ-ray absorption, making them suitable for shielding against ionizing radiation [14]. The mechanism underlying ZFN's protective ability includes photoelectric absorption, Compton scattering, and pair production, all of which contribute to attenuating γ-ray intensity [15,16]. Moreover, ZFN-based materials have been identified as efficient dosimetric agents, capable of absorbing hazardous radiation [17]. Another promising radioprotective compound is folic acid (FA), also known as vitamin B9, which plays a key role in DNA replication, amino acid metabolism, and enzymatic one-carbon transfer reactions [18,19]. FA is a crucial cofactor for various biochemical processes, including the remethylation of homocysteine (HCY) into methionine, a reaction essential for maintaining cellular function [20]. Additionally, FA exhibits potent antioxidant activity, scavenging ROS, modulating lipid metabolism, and restoring antioxidant enzyme activity, thereby reducing oxidative stress [21]. Recent studies have shown that FA-functionalized magnetic iron oxide nanoparticles (Fe3O4-FA) significantly alleviate DNA damage and regulate lipid metabolism in γ-irradiated models, suggesting their therapeutic potential in radiation protection [6,22]. In light of these findings, this study aims to develop a novel nanocomposite-based radioprotective agent, FA/ZFN/CYS, that integrates the antioxidant properties of CYS, the shielding ability of ZFN, and the DNA-protective effects of FA. The physicochemical characteristics of FA/ZFN/CYS were analyzed using SEM, TEM, XRD, FTIR, EDX, and zeta potential analysis. Furthermore, its radioprotective efficacy was assessed through biochemical markers of liver and kidney function, inflammatory cytokine levels, antioxidant enzyme activity, and histopathological changes in male Wistar rats exposed to γ-irradiation. The proposed mechanism of FA/ZFN/CYS nanocomposite for radioprotection is displayed in Scheme 1.