The gut microbiome is widely analyzed using high-throughput sequencing, such as 16S rRNA gene amplicon sequencing and shotgun metagenomic sequencing (SMS). DNA extraction is known to have a large impact on the metagenomic analyses. The aim of this study was to compare DNA extraction protocols for 16S sequencing. In that context, four commonly used DNA extraction methods were compared for the analysis of the gut microbiota. Commercial versions were evaluated against modified protocols using a stool preprocessing device (SPD, bioMérieux) upstream DNA extraction. Stool samples from nine healthy volunteers and nine patients with aClostridium difficileinfection were extracted with all protocols and 16S sequenced. Protocols were ranked using wet- and dry-lab criteria, including quality controls of the extracted genomic DNA,alpha-diversity, accuracy using a mockcommunity of known composition and repeatability across technical replicates. SPD improved overall efficiency of three of the four tested protocols compared with their commercial version, in terms of DNA extraction yield, samplealpha-diversity, and recovery of Gram-positive bacteria. The best overall performance was obtained for the S-DQ protocol, SPD combined with the DNeasy PowerLyser PowerSoil protocol from QIAGEN. Based on this evaluation, we strongly believe that the use of such stool preprocessing device improves both the standardization and the quality of the DNA extraction in the human gut microbiome studies.