The gut microbiota has long been of research interest due to its importance to human health, nutrition, and well-being. A healthy gut microbiota (properly balanced bacterial groups) is normally required for human health by maintaining host immune homeostasis, nutrient intake, as well as gut development. An unbalanced gut microbiota is often associated with the pathogenesis of both acute and chronic diseases, such as infectious diarrhea, inflammatory bowel diseases, obesity, diabetes, colon cancer, and neonatal necrotizing enterocolitis. It has been clearly demonstrated that the gut microbiota can be modulated by many factors including diet. Dietary fiber can benefit human health through different mechanisms, including physical effect, immunomodulation, colonic fermentation, and prebiotic effect. In the past, determination of microbiota changes and corresponding functions in response to dietary treatments has been a challenge, largely due to the limitation of conventional microbiological technology. With the completion of human and other genome projects as well as the ongoing human microbiome project, revolutionary technologies characterized by culture-independent, high throughput, high efficiency and rapid computation are becoming available for studying gut microbiota. These advanced tools include quantitative polymerase chain reaction (Q-PCR) analysis, PCR-based DNA profiling techniques, DNA microarray, flow cytometry, insertion sequencing, and particularly next generation DNA sequencing and bioinformatics analyses. These robust, powerful methods have enabled more comprehensive studies on the gut microbiota and its functions including its interaction with host and diet. Moreover, integrative use of gnotobiotic and knock-out/transgenic animals and functional genomics approaches would further improve our understanding of the gut microbiome. The advances in microbiota research would particularly enhance our ability to study dietary fiber and its benefits to human health and nutrition. New knowledge on the modulation of gut microbiota by dietary fiber is critical for the development of effective strategies to improve human health and to treat microbiota associated-diseases.