Low temperature causes injuries to plants during winter, thereby it affects kiwi fruit quality and yield. However, the changes in metabolites and gene expression during cold acclimation (CA) and deacclimation (DA) in kiwi fruit remain largely unknown. In this study, freezing tolerance, carbohydrate metabolism, and β-amylase gene expression in two Actinidia arguta cv. “CJ-1” and “RB-3” were detected from CA to DA stages. In all acclimation stages, the “CJ-1” was hardier than “RB-3” and possessed lower semi-lethal temperature (LT50). Furthermore, “CJ-1” had a more rapid acclimation speed than “RB-3”. Changes of starch, β-amylase, and soluble sugars were associated with freezing tolerance in both cultivars. Starch contents continued to follow a declining trend, while soluble sugars contents continuously accumulated in both cultivars during CA stages (from October to January). To investigate the possible molecular mechanism underlying cold response in A. arguta, in total, 16 AcBAMs genes for β-amylase were identified in the kiwi fruit genome. We carried out localization of chromosome, gene structure, the conserved motif, and the analysis of events in the duplication of genes from AcBAMs. Finally, a strong candidate gene named AaBAM3 from AcBAMs was cloned in Actinidia arguta (A. arguta), The real-time qPCR showed that AaBAM3 gene expression in seasonal changes was consistent with changes of soluble sugars. These results reveal that AaBAM3 may enhance the freezing tolerance of A. arguta through increasing soluble sugar content.