Seed vigor of tobacco (Nicotiana tabacum L.) is established during seed development stage, while its regulators remain largely unknown. Here, a comparative peptidomics analysis of the developing seeds was conducted to reveal the regulators involving the establishment of tobacco seed vigor. The most significant difference of seed vigor was observed between seeds harvested at 20 and 30 days after pollination (DAP), and then the corresponding seeds were collected separately for peptidomics analysis. A total of 2932 and 2812 nonredundant peptides were identified in seeds harvested at 20 and 30 DAP, respectively. In which, 349 differentially expressed peptides (DEPs) were characterized. To explore the potential functions of these DEPs, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were further analyzed according to their precursor proteins. Most DEP precursor proteins were involved in response to abiotic stimulus, response to water, and protein processing in endoplasmic reticulum. Further, the peptides derived from the precursor proteins, such as late embryogenesis abundant (LEA) protein, heat shock protein, peroxiredoxin, and globulin proteins, may regulates the establishment of seed vigor by influencing reactive oxygen species. The results provide a foundation for further exploration of the peptides functions on the establishment of seed vigor in tobacco.