Analysis of the human gut microbiome, collective genomes of over 100 trillion cells of intestinal microbes which form the complex bacterial community, has recently become more practical due to remarkable advances in next-generation sequencing technologies (NGS). Several studies using NGS-based metagenomic approaches have been conducted to comprehensively analyze genes/functions and species composition in the human gut microbiome. These NGS-based approaches have demonstrated that their ecological and biological features that have been rather difficult to pursue can now be characterized with relative ease and high-throughput. There have also been several analytical developments and improvements to accurately evaluate the microbiome data from viewpoint of biology and ecology. In addition to the impact of external factors such as diet, these NGS-based studies have strongly suggested that the human gut microbiome has profound influences on various host physiologies including disease, of which the gut microbiome exhibited ecologically and functionally aberrant structure as compared with that of healthy control. On the other hand, the detailed assessment of the effect of experimental protocols including sample storage and delivery, DNA preparation, and sequencers used on the gut microbiome structure is also necessary. I herein present the NGS-based methodology for the human gut microbiome analysis.