AbstractBackgroundMost of the previous studies have demonstrated the potential antidepressive and anxiolytic role of prebiotic supplement in male subjects, yet few have females enrolled. Herein, we explored whether prebiotics administration during chronic stress prevented depression‐like and anxiety‐like behavior in a sex‐specific manner and the mechanism of behavioral differences caused by sex.MethodsFemale and male C57 BL/J mice on normal diet were supplemented with or without a combination of fructo‐oligosaccharides (FOS) and galacto‐oligosaccharides (GOS) during 3‐ and 4‐week chronic restraint stress (CRS) treatment, respectively. C57 BL/J mice on normal diet without CRS were used as controls. Behavior consequences, gut microbiota, dysfunction of gut and brain–blood barriers, and inflammatory profiles were measured.ResultsIn the 3rd week, FOS + GOS administration attenuated stress‐induced anxiety‐like behavior in female, but not in male mice, and the anxiolytic effects in males were observed until the 4th week. However, protective effects of prebiotics on CRS‐induced depression were not observed. Changes in the gene expression of tight junction proteins in the distal colon and hippocampus, and decreased number of colon goblet cells following CRS were restored by prebiotics only in females. In both female and male mice, prebiotics alleviated stress‐induced BBB dysfunction and elevation in pro‐inflammatory cytokines levels, and modulated gut microbiota caused by stress. Furthermore, correlation analysis revealed that anxiety‐like behaviors were significantly correlated with levels of pro‐inflammatory cytokines and gene expression of tight junction proteins in the hippocampus of female mice, and the abundance of specific gut microbes was also correlated with anxiety‐like behaviors, pro‐inflammatory cytokines, and gene expression of tight junction proteins in the hippocampus of female mice.ConclusionFemale mice were more vulnerable to stress and prebiotics than males. The gut microbiota, gut and blood–brain barrier, and inflammatory response may mediate the protective effects of prebiotics on anxiety‐like behaviors in female mice.