Endoreplication is particularly important in the context of silk protein synthesis within the silk gland cells of silkworms. Our previous research indicated that the BmE2F1 enhances the silk yield of silkworm cocoons, but the underlying molecular mechanism remains elusive. In this study, we employed RNA-sequencing to dissect the transcriptional profiles of silk glands in the wild-type Dazao silkworm strain and the overexpression (OE) silkworm strain with specific overexpression of the BmE2F1 gene in silk glands. Among the 1126 differentially expressed genes (DEGs), many related to DNA replication (endoreplication in silk glands of silkworm larvae) were significantly enriched. Quantitative real-time PCR confirmed that overexpression of BmE2F1 led to a substantial increase in the expression of 13 genes involved in the DNA replication pathway. Additionally, BmE2F1 upregulated the expression of BmCyclin E, a pivotal gene in the G/S phase transition. Moreover, BmE2F1 overexpression in silk glands significantly boosted DNA replication and concurrently increased the DNA content within silk glands. In conclusion, BmE2F1 regulates endoreplication in silk gland cells of silkworms through dual mechanisms: firstly, by enhancing the formation of the DNA replication complex
and secondly, by facilitating the cells' entry into the S phase.