Abstract:[Objective] Giant panda is one of the first-class protected animals in China, which the population is facing continuous threats such as infectious diseases and habitat fragmentation. Bacterial infection and imbalance of bacterial community in the genitourinary system of giant panda could affect the reproductive health, in severe cases, it can also lead to abortion, which is one of the causes of reproductive disorders in giant panda. In this study, our aim was to examine the bacterial community composition of semen and prepuce secretion and isolate potential pathogenic bacteria of giant panda.[Methods] 13 of prepuce secretions and 12 of semen samples of giant panda were used to determine the bacteria species by 16S rRNA amplicon sequencing technique, bacterial separation and PCR methods.[Results] The results showed that the abundance of Firmicutes was the highest in the samples of prepuce and semen of giant panda at phylum level. Moreover, at the genus level, the bacterial community composition of prepuce was changed in different periods, Corynebacterium and Dolosicoccus were the most abundant microbiome in preputial samples at phase I with relative abundances of 15.45% and 12.40%, respectively. Streptococcus and Escherichia were the most abundant microbiome in preputial samples at phase II, with relative abundance of 37.94% and 9.68%, respectively. Bacteroides and Prevotella were the most abundant microbiome in seminal samples, with relative abundance of 14.40% and 12.88%, respectively. The results of bacterial diversity showed that the diversity in seminal samples was higher than that in preputial samples; there were no significant difference between preputial samples at stages I and stage II. Klebsiella pneumonia and other potential pathogenic bacteria were obtained by bacterial isolation and culture.[Conclusion]] In this study, we analyzed the bacterial community composition of the prepuce secretions in different periods and semen of male giant pandas, and the dominant microbiome were different. It was hypothesized that potential pathogenic bacteria were in the prepuce and semen of giant pandas, which may pose a threat to the reproductive system health of giant panda, and the pathogenicity needs further research.