Metagenomic analysis based on the 16S rRNA gene is generally performed to examine the diversity and abundance of commensal bacteria in feces, which is now recognized to be associated with human health and diseases. Guanidine thiocyanate (GuSCN) solution is used as a less onerous way compared with a frozen method to transport and stock fecal samples at room temperature for DNA analysis; however, optimal methods to measure fecal bacterial composition in GuSCN solution remain to be investigated. Here, we examined the influence of various factors such as pretreatment (e.g., removing GuSCN solution and washing feces with phosphate-buffered saline (PBS) before mechanical lysis), fecal concentration in the GuSCN solution, storage time, and position of fecal subsampling on the 16S rRNA-based analysis of fecal bacteria in GuSCN solution. We found that pretreatment and fecal concentration affected the bacterial composition, and a little change was noted with subsampling position. Based on these results, we propose a basic protocol, including fecal sampling, sample storage, and DNA extraction, for the 16S rRNA-based analysis of bacterial composition in feces suspended in GuSCN solution.