Abstract: To reveal the molecular mechanism of seed photodormancy and after-ripening in tobacco, wild-type (WT), NtPHYB1 over expression (NtPHYB1-OE) and deficient (NtPHYB1-RNAi) tobacco seeds were used in this study, in conducting after-ripening experiments in two environments with constant or natural temperature. The results showed that NtPHYB1-RNAi fresh seeds had a significant increase in photodormant level compared with WT fresh seeds; However, the photodormancy of NtPHYB1-OE fresh seeds did not show significant change. The after-ripening of WT and NtPHYB1-OE seeds was accelerated under variable temperature compared with under constant temperature storage, while that of NtPHYB1-RNAi seeds was not significantly changed. Compared with WT fresh seeds, the expression of NtABI3 in NtPHYB1-RNAi fresh seeds was significantly increased, while the expression of NtABI5 in NtPHYB1-OE fresh seeds was significantly decreased. In WT seeds, expression of NtNCED6 and NtGA2ox2 decreased slowly when after-ripening under variable temperature, while NtCYP707A1 and NtGA3ox2 were up-regulated slowly. In NtPHYB1-RNAi seeds, NtNCED6 expression decreased slowly then, while NtCYP707A1 expression increased slowly. In NtPHYB1-OE seeds, NtNCED6 expression decreased slowly then, while expression of NtCYP707A1, NtGA3ox2, NtGA2ox2 and NtGAI decreased faster. In conclusion, NtPHYB1 interacts with storage temperature to regulate photodormancy and after-ripening by regulating expressions of genes involved in ABA and GA signal and metabolism in tobacco seeds.