Abstract: To investigate the response of tobacco seed germination process to low temperature stress, the seeds germinated for 1, 2 and 3 days were treated with low temperature for 2 days (samples were named LT1, LT2 and LT3 respectively), and the changes of proteins and polypeptides in seeds exposed to low temperature at different stages of germination were analyzed. The seeds germinated for 2, 3 and 4 days were used as control (samples were named GS2, GS3 and GS4 respectively), forming three combinations of treatment GS2 vs LT1, GS3 vs LT2 and GS4 vs LT3. The results showed that the intersection of differentially expressed protein of GS2 vs. LT1, GS3 vs. LT2 and GS4 vs. LT3 had a total of 20 polypeptides. The results of GO and KEGG enrichment analysis indicated that the differential polypeptides of the three combinations were related to carbon metabolism and amino acid biosynthesis. In addition, eight peptides were identified to be involved in low temperature response, and the peptide Nitab4.5_0002257g0090 was identified in three combinations at the same time, suggesting that it may have an important function in low temperature response. This study revealed the responsiveness of tobacco seeds to low temperature signaling pathways, and provided a data basis for the subsequent mining of peptides in the low temperature response process.