The E2 promoter binding factors (E2Fs) are a group of transcriptional regulators that govern the cell cycle and play crucial roles in various cellular physiological processes, including proliferation and embryonic development. In this study, we identified four homologous genes-Lr-E2F3, Lr-E2F4, Lr-E2F5, and Lr-E2F8-from the lamprey (Lethenteron reissneri) genome database. Phylogenetic tree analysis was conducted to elucidate the evolutionary relationships within the E2F family across different species. Furthermore, analyses of motifs, domains, gene structures, and 3D structures reinforced the conservation of the E2F family. Notably, synteny analysis revealed that the neighboring genes of the Lr-E2Fs exhibited greater diversity compared to those in jawed vertebrates. Activity assays indicated that Lr-E2Fs may be involved in lamprey innate immunity mediated by NF-кB. Additionally, morphological observations of embryos microinjected with Cas9/sgRNA demonstrated that E2F-deficient lamprey embryos displayed embryonic lethality, suggesting that Lr-E2Fs play a significant role in lamprey embryonic development. In summary, our research not only provides new insights into the evolution of Lr-E2Fs but also offers valuable clues regarding their functional roles.