Abstract Dysbiosis of gut microbiota exists in the pathogenesis of many autoimmune diseases, including lupus. Currently, women with lupus are recommended to avoid pregnancy, as lupus patients who experienced pregnancy usually had more severe disease flares. However, the possible role of gut microbiota in the link between pregnancy and exacerbation of lupus remains to be explored. In the present study, in the classical lupus mouse model MRL/lpr, we compared the structures of gut microbiota in pregnant and lactating individuals vs. age-matched naïve mice. Consistent with studies on non-lupus mice, both pregnancy and lactation significantly changed the composition and diversity of gut microbiota. Strikingly, modulation of gut microbiota using the same strategy resulted in different disease outcomes in experienced (which had undergone pregnancy and lactation) vs. control (naïve) MRL/lpr females: while vancomycin treatment attenuated lupus in naïve mice, it even exacerbated lupus in experienced mice. L. animalis flourished in the gut upon vancomycin treatment, and direct administration of the bacteria recapitulated the differential effects of vancomycin in experienced vs. control mice. A pregnancy-related enzyme, DHNA, was significantly inhibited by L. animalis; however, this inhibition was only apparent in experienced mice, which explained, at least partially, the lack of beneficial response to vancomycin in these mice. The differential production of IL-10 and IFNγ in experienced vs. control mice further explained why the disease phenotypes varied between the two types of mice bearing the same treatment. These results suggest that pregnancy and lactation interfere with the response of autoimmunity to modulation of gut microbiota.