High throughput sequencing of gut microbiota helps to understand the nutrition and health of the host. In this sense, it is an important tool for improving wildlife conservation and management. Little is known about the microbial communities occupying the gastrointestinal tract and the differences of microbiota among primate species. We conducted a microbiome study of the gastrointestinal tract in golden snub-nosed monkey (Rhinopithecus roxellanae), green monkey (Chlorocebus aethiops sabaeus), and ring-tailed lemur (Lemur catta), using the 16S rRNA gene amplicon sequencing and shotgun metagenomic sequencing. The gastrointestinal tract of all three primates were characterized by Firmicutes, Bacteroidetes, and Proteobacteria, although the relative abundance of these taxa differed across non-human primate species and gastrointestinal segments. In addition, the stomach of the golden monkey demonstrated a strong signal of microbial metabolic activity, especially in metabolizing carbohydrate. Prevotella multisaccharivorax and Selenomonas bovis were the main contributors to the function, and the enzymes involved in these metabolic activities were mainly derived from the glycoside hydrolase (GH) family. Microbial composition and function vary widely across non-human primate species and gastrointestinal segments. Therefore, while faecal samples are necessary for studying many mammals, particularly in the wild, data from the gastrointestinal tract have the potential to greatly improve our knowledge of host-gut microbe interactions. This information can contribute to the development of conservation interventions and animal care protocols in the wild and captivity to more fully describe a species’ nutritional needs and advance health and survival.