In previous studies, we found young plants propagated from acclimated D. odorifera T. Chen trees displayed increased cold tolerance. To understand the molecular and physiological mechanisms underlying this phenomenon, we prepared acclimated and non-acclimated young D. odorifera T. Chen trees for physiological, RNA-Seq, and IsoSeq analyses. First, both the acclimated and non-acclimated young trees were grown in 10 ℃ condition, then treated at -3 ℃ for one day, and finally recovered at 10 ℃. The measurement of physiological parameters, including superoxide dismutase, peroxidase, malondialdehyde, thiobarbituric acid, and soluble sugar, showed that the physiological change of acclimated plants is smoother than non-acclimated plants. The RNA-Seq analyses pointed out that cold acclimation already fixed the different gene expression patterns of D. odorifera T. Chen trees. The hormone-related, secondary metabolic, and signal transduction related biological genes tend to show different expressions between the acclimated and non-acclimated D. odorifera T. Chen trees. Moreover, the change of gene expression for some biological processes, such as alpha-Linolenic acid metabolism and its response to hydrogen peroxide, seems to occur earlier in non-acclimated than acclimated plants. The ISOSeq analyses pointed out that alternative splicing (AS) of some genes was also found, and these AS events were predicted to play important roles in regulating different expression patterns between non-acclimated and acclimated plants. Therefore, according to the performance of this study, we are able to provide some novel understanding of cold tolerance enhanced by cold acclimation in perennial trees.