Actinidia arguta (kiwiberry) is a perennial deciduous vine that has a very strong overwintering ability. We speculated that trehalose metabolism, which plays a pivotal role in the stress tolerance of plants, may be involved in the cold acclimatization of A. arguta. Transcriptome analysis showed that the expression of AaTPPA, encoding a trehalose-6-phosphate phosphatase (TPP), was upregulated in response to low temperature. AaTPPA expression levels were much higher in lateral buds, roots, and stem cambium than in leaves in autumn. In AaTPPA-overexpressing (OE) Arabidopsis thaliana, trehalose levels were 8 ~ 11 times higher than that of the wild type (WT) and showed different phenotypic characteristics from WT or overexpression lines of OtsB, the E. coli TPP gene. Surprisingly, AaTPPA-OE A. thaliana had significantly higher freezing tolerance than WT and OtsB-OE lines. Transient overexpression of AaTPPA in A. arguta leaves increased ROS scavenging capacity and the contents of soluble sugars and proline. AaERF64, an ethylene-responsive transcription factor, was induced by ethylene treatment, and bound to the GCC-box of the AaTPPA promoter to activate its expression. AaTPPA expression was also induced by abscisic acid (ABA). Overall, the temperature decrease in autumn induces AaERF64 expression through the ethylene signal transduction pathway, which in turn upregulates AaTPPA expression, leading to the accumulation of osmotic protectants including soluble sugars and proline in the overwintering tissues of A. arguta.