High-quality and high-throughput sequencing technologies are required for therapeutic and diagnostic analyses of human gut microbiota. Here, we evaluated the advantages and disadvantages of the various commercial sequencing platforms for studying human gut microbiota. We generated fecal bacterial sequences from 170 Korean subjects using the GS FLX+(V1–4), Illumina MiSeq (V1–3, V3–4 and V4), and PacBio (V1–9) systems. Comparative analyses revealed that the PacBio data showed the weakest relationship with the reference whole-metagenome shotgun datasets. The PacBio system generated sequences with a significantly higher level of deletions than datasets generated by other platforms, with an abnormally high proportion of sequences assigned to the phylum Proteobacteria. Low sequencing accuracy and low coverage of terminal regions in public 16 S rRNA databases deteriorate the advantages of long read length, resulting in low taxonomic resolution in amplicon sequencing of human gut microbiota.