The Orf virus (ORFV) poses a significant threat to livestock and human health, causing economic losses in the livestock industry and potential zoonotic infections. Given the limitations of current vaccines, the objective of this study was to investigate the immune response and gut microbiota modulation induced by the ORFV B2L gene-based DNA vaccine (GV) and the live attenuated vaccine (LV) in rats. The findings of this study will provide a scientific foundation for the development of more effective vaccines. Female Sprague-Dawley rats, which were free of specific pathogens, were divided into three groups. The experiment included three groups: the first group was designated as the GV group, the second group was designated as the LV group, and the third group was designated as the control group. Rats in the GV group received intra-muscular injection of 100μg/dose of pVAX - B2L - asd plasmid, those in the LV group were immunized with a commercial live - attenuated vaccine, and the control group was injected with PBS. After immunization, various immune - related parameters, such as T - cell subsets, antibody levels, cytokines, and oxidative stress markers, were measured. To this end, composition and function of gut microbiota were thoroughly examined through the implementation of 16S rRNA gene sequencing and PICRUSt-2 functional prediction. The GV group exhibited elevated levels of cellular and humoral immunity. It had a higher percentage of CD4 and CD8 T cells, enhanced levels of cytokines i.e. IL - 2, IL - 6, and TNF - α, elevated IgA, IgG antibody production compared to the LV and control groups. Additionally, the GV group showed reduced oxidative stress. In terms of gut microbiota, GV immunization led to an increase in beneficial bacteria like Lachnospi-raceae_NK4A136_group and a decrease in harmful or potentially pathogenic bacteria. KEGG pathway analysis indicated that differential flora exhibited an increase in metabolic pathway diversity, including those related to biological systems, metabolism, and human diseases. In sum, the results of the present study demonstrate that the ORFV B2L DNA vaccine (GV) elicited a more robust immune response and exerted a beneficial effect on composition and function of the gut microbiota compared with ORF live-attenuated vaccine. The results of the present study indicate that modulation of gut microbiota by GV vaccine play a crucial role in enhancing vaccine efficacy. The current study provides new perspectives on ORFV vaccine development and its association with vaccines and gut microbiota modulation.