Objective To investigate the characteristics of gut microbiota in rhesus macaques of different ages.Methods Stool samples were collected from 33 adult (5-10 years old) and 17 old (>10 years old) rhesus macaques and the V3 region of the 16S rDNA gene was examined by Illumina Miseq high-throughput sequencing. The abundance and composition of the gut microbiota were analyzed by cluster analysis. Results DNA sequence analysis was successfully performed ( P >0. 05). The Chao1 ( P =0. 0174), Simpson ( P =0. 0258), and ACE ( P =0. 0121) indexes were lower and the Shannon index ( P = 0. 0132) was higher in the old group than the adult group. At the phylum level, the relative abundance of Bacteroidetes was higher in the old group than the adult group ( P = 0. 013). Conversely, the relative abundance of Firmicutes, Spirochaetes, Verrucomicrobia and Lentisphaerae was lower in the old group than the adult group ( P =0. 0283, P = 0. 0002, P = 0. 0482, P = 0. 0242). At the family level, the relative abundance of Prevotellaceae was higher in the old group than the adult group ( P = 0. 0001), while the relative abundance of Ruminococcaceae,Clostridiales, Spirochaetaceae, and Christensenellaceae was lower in the old group ( P =0. 0039, P =0. 0080, P =0. 0002, P =0. 0021). At the genus level, the relative abundance of unidentified Prevotellaceae was higher in the old group than the adult group ( P =0. 0001). By contrast, the relative abundance of Lactobacillus, Sarcina and unidentified Spirochaetaceae was lower in the old group than the adult group ( P = 0. 0114, P = 0. 0227, P = 0. 0028). β-diversity analysis showed that the adult and old groups were distributed in different quadrants, and there was a significant difference between the two groups ( P =0. 003). LDA effect size analysis indicated that Streptococcus, Buchnera, and Lactobacillus were biomarkers at the genus level in the adult group. Conclusions Rhesus macaques have a lower abundance and higher diversity of gut microbiota with increasing age.