Loquat (Eriobotrya japonica) is an evergreen fruit tree native to China, with a flowering period that typically occurs in winter (October to January), making it vulnerable to low-temperature stress during critical reproductive stages. However, the molecular mechanisms underlying cold tolerance in loquat remain largely unclear. In this study, transcriptome data from multiple loquat cultivars were analyzed using Weighted Gene Co-expression Network Analysis (WGCNA), identifying two gene modules (brown and turquoise modules) highly associated with cold treatment. Among the cold-responsive candidates, the Rab5 family GTPase EjRabF2b was consistently upregulated under low-temperature conditions. Functional validation revealed that overexpression of EjRabF2b in Arabidopsis thaliana and tomato significantly enhanced cold tolerance, while its silencing in loquat compromised stress resistance. Mechanistically, EjRabF2b contributed to maintaining cell membrane integrity and enhancing antioxidant enzyme activity. Promoter analysis and interaction assays further confirmed that the C2H2-type transcription factor EjZAT10 directly binds to the promoter of EjRabF2b and activates its transcription under cold stress. Collectively, this study uncovers a regulatory module composed of EjZAT10 and EjRabF2b that participates in loquat cold adaptation through vesicle-mediated antioxidant defense and membrane protection, offering a theoretical foundation and potential targets for the molecular breeding of cold-tolerant cultivars.