Objective To explore the antidepressant effects of acupuncture at Baihui and Zusanli acupoints on mice with chronic unpredictable mild stress(CUMS)-induced depressive-like behavior and its association with changes in gut microbiota.Methods Sixty mice were randomly divided into four groups:control group(Ctrl),control+acupuncture group(Ctrl+AP),CUMS group,and CUMS+acupuncture group(CUMS+AP).The CUMS and CUMS+AP groups underwent 6-week CUMS treatment.During the last three weeks of CUMS,mice in the Ctrl+AP and CUMS+AP groups received 20 minutes of acupuncture at Baihui and Zusanli acupoints daily.Sucrose preference test and tail suspension test were used to assess the depressive-like behavior in mice.After the behavioral tests,fecal samples were collected from the mice,and the gut microbiota composition was analyzed using 16S rRNA gene sequencing.Results Acupuncture alone had no effect on sucrose preference or tail suspension test results in mice(P＞0.05).CUMS induced depressive-like behavior in mice,as evidenced by decreased sucrose preference(P＜0.001)and increased immobility time in the tail suspension test(P=0.003).The CUMS+AP group showed increased sucrose preference(P=0.035)and decreased immobility time in the tail suspension test(P=0.045)compared to the CUMS group.Acupuncture and CUMS did not alter the richness of gut microbiota in mice(P＞0.05),but they caused significant changes in gut microbiota composition(P=0.002).CUMS increased the abundance of six bacterial genera,including Patulibacter,and decreased the abundance of 26 bacterial genera,including Faecalibacterium(P＜0.05).The CUMS+AP group showed increased abundance of 10 bacterial genera,including Faecalibacterium,and decreased abundance of four bacterial genera,including Patulibacter,compared to the CUMS group(P＜0.05).Each group of mice had specific gut microbiota.The CUMS group showed significant enrichment of gene functions related to retinol metabolism,ubiquitin system,N-glycan biosynthesis,steroid hormone biosynthesis,apoptosis,and thermogenesis compared to the control group(P＜0.05),while acupuncture reduced the enrichment of these gene functions in the CUMS+AP mice(P＜0.05).Conclusion Acupuncture at Baihui and Zusanli acupoints has antidepressant effects and can partially restore the depression-induced dysbiosis of gut microbiota.Acupuncture at Baihui and Zusanli acupoints may exert antidepressant effects by influencing gut microbiota composition and metabolism functions of retinol,ubiquitin,N-glycan,and steroids.