Rapeseed is a typical winter crop, and its freezing stress tolerance is a major feature for winter survival. Therefore, it is important to comprehend clearly the physical and molecular mechanisms of rapeseed under freezing stress conditions. This study investigates the physical and transcriptome changes of two rapeseed lines, ‘J8634-B-30’ and ‘EMS26’, under cold acclimation and freezing temperature treatments. The proline content of ‘J8634-B-30’ at 5 °C increased 8.7-fold compared to that before treatment, and there was no significant change in that of ‘EMS26’ RNA-sequencing analysis revealed 5,083 differentially expressed genes (DEGs) of ‘J8634-B-30’ under cold acclimation condition. Among the genes, 2,784 (54.8%) were up-regulated and 2,299 (45.2%) were down-regulated. The DEGs of ‘EMS26’ under cold acclimation condition were 5,831 genes, and contained 2,199 up-regulated genes (37.7%) and 3,632 down-regulated genes (62.3%). Among them, only DEGs annotated in the cold response-related signaling pathways were selected, and their expression in the two rapeseed lines was compared. Comparative DEGs analysis indicated that cold response related signaling pathways are proline metabolism and ABA (Abscisic acid) signaling. And ICE (Inducer of CBF expression) - CBF (C-repeat-binding factor) - COR (Cold-regulated) signaling were the significantly differentially expressed transcripts in the two rapeseed lines. The major induced transcripts of ‘J8634-B-30’ induced P5CS (Δ‘-pyrroline-5-carboxylate synthetase), which is related to proline biosynthesis, PYL (pyrabactin resistance-like protein, ABA receptor) and COR413 (cold-regulated 413 plasma membrane 1). In conclusion, these result provide a foundation for understanding the mechanisms of freezing stress tolerance in rapeseeds. Further functional studies should be performed on the freezing stress-related genes identified in this study, which can contribute to the transgenic and molecular breeding for freezing stress tolerance in rapeseed.