Abstract Background The number of large-scale studies on the gut microbiota in human cohorts is rapidly increasing. However, the few and expensive options for storage of fecal samples at room temperature have been an obstacle for large-scale metagenomic studies and the development of clinical/commercial personal metagenomic sequencing. Results In this study, we systematically tested a novel N-octylpyridinium bromide-based fecal sample preservation method and compared it with other currently used storage methods. We found that the N-octylpyridinium bromide-based method enabled preservation of the bacterial composition in fecal samples transported and stored at room temperature for up to at least 14 days. Conclusions We describe a novel chemical stabilizer that allows cost-effective transportation and storage at room temperature for several days with preservation of bacterial composition. This method will facilitate sample collection even in remote area and also enable transport via normal commercial transportation routes.