Abstract:The gut harbors the microbiota with complex structure and diverse functions, which plays an important role in host immunity, nutrient absorption, and metabolic regulation. The rapid development of sequencing technologies such as 16S rRNA gene sequencing and metagenomic sequencing has generated massive data of gut microbiota, among which many unassembled sequences are considered as the microbial dark matter. Through the combination of a variety of culture methods and high-throughput sequencing, a large number of microorganisms have been isolated from the guts of human, mouse, and pig in recent years, which has significantly enriched the bacterial strain resources and provided a basis for the analysis of microbial dark matter and the research on the functions and application of gut microbiota. Although the culturability of microorganisms is affected by many factors and most microorganisms are still uncultured, the acquisition of microbial resources is indispensable for the study of etiology and the analysis of physiological and genetic characteristics of bacterial strains. The isolation and culture of gut microorganisms are of great significance for the deep research on gut microbiota from association study to functional verification, causality elucidation, and functional strain development. This article summarized the factors that affect the culturability of microorganisms, introduced the culture methods of gut microorganisms, and reviewed the progress and application of gut microorganism culture, aiming to give new insights into this field.