Cold stress severely impairs plant growth, development, and yields. L-ascorbic acid (ASA), a crucial antioxidant, is pivotal in mitigating stress-induced damage. Previous research found that 2-keto-L-gulonic acid (2KGA), a precursor of ASA in its industrial production, effectively enhances the endogenous ASA content in plants. We hypothesized that 2KGA might alleviate chilling stress and tried to verify it through a cultivation experiment of Arabidopsis thaliana. The results demonstrate that the application of 2KGA significantly increased ASA content (24.58%) and up-regulated ASA biosynthetic genes in Arabidopsis at 4 °C for 24 h. Furthermore, 2KGA alleviated the decrease in fresh weight (17.05%) and total chlorophyll content (15.85%) caused by low temperatures. The contents of proline, soluble sugar (SS), soluble protein (SP), and the activities of antioxidant enzymes including superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) were significantly increased under the 2KGA treatment at low temperatures, while the malondialdehyde (MDA) content was reduced. Moreover, 2KGA up-regulated the ICE-CBF-COR signaling pathway in response to cold stress. These collective findings strongly support the involvement of 2KGA in enhancing cold tolerance in Arabidopsis, presenting an innovative approach for agricultural practices aimed at enhancing crop resilience to environmental stresses.