<p>During postharvest cold storage, chilling-sensitive fruits and vegetables often develop chilling injury (CI) due to low-temperature stress, resulting in quality deterioration and a shortened shelf life. Glycine betaine (N, N, N-trimethylglycine; GB), a natural osmoprotectant, can effectively suppress CI symptoms and maintain the postharvest quality of fruits and vegetables through multifaceted regulatory mechanisms. This paper systematically reviews GB extraction techniques, its synthesis and metabolism in fruits and vegetables, the pre- and post-harvest application methods for GB in preservation, and its metabolic regulatory roles in pre- and post-harvest preservation. Research has demonstrated that exogenous GB treatment significantly delays postharvest ripening and senescence by inhibiting ethylene biosynthesis and abnormal respiratory metabolism, modulating the reactive oxygen species (ROS) scavenging system and antioxidant enzyme activities, preserving cell wall constituents, and maintaining cell membrane integrity through its osmoprotective function. Concurrently, GB effectively mitigates low-temperature stress-induced increases in membrane permeability, lipid peroxidation, and cellular energy imbalance by stabilizing membrane lipid structures, improving energy metabolism homeostasis, enhancing phenylpropanoid pathway activity, and regulating the balance of sugar and amino acid metabolism. Furthermore, the synergistic application of GB with technologies such as nanomaterials, chitosan coatings, and heat treatment can further enhance its preservation efficacy. This review reveals the potential of GB as an alternative to synthetic preservatives, providing a theoretical basis and technical strategies for reducing postharvest losses and extending the storage duration of fruits and vegetables.</p>