Gut microbiota is closely related to human health, and its composition can give us health information. The large-scale population sampling is required on gut microbiome research; however, fresh feces samples are not easy to obtain, and rapid low-temperature freezing is difficult to achieve. With the development of technology, preservation solutions are widely used for sample collection, storage, and transport under normal temperature conditions. Preservation solutions can be used in large scale sample collection, wide geographical distribution, diverse on-site sampling conditions, heavy workload, and poor transportation conditions. In this study, five healthy volunteers were recruited. After collecting their fresh stool samples, effect of 5 different commercial preservation solutions was evaluated at room temperature. Samples in different preservation solutions after placing fresh stool samples at the 0, 1, 3, 7, 15, and 30 days were collected. All samples were tested by 16S rRNA V3–V4 high-throughput sequencing to analyze the influence of microbiome composition in different preservation solutions. The results show that different preservation solutions had distinct effects on the gut microbiome composition. Compared with the control, different preservation solutions had little effect on the amount of OUTs; preservation solutions A, B and C were closer to the control in the composition of the gut microbiota, but preservation solution D significantly changed the composition by increasing Actinobacteria and Firmicutes abundance. With the time, all solutions tended to reduce the diversity of the microbiota. Preservation solution E significantly reduced the diversity of the flora; on the 30th day, all five solutions changed the composition; the individual differences in the composition of the gut microbiome were the main factors affecting the similarity of each sample, and were derived from different stools donors. The same samples, no matter which storage solution and storage time, were directly closer to each other. Different storage solutions had different effects on the content of Gram-positive bacilli, Gram-positive cocci and Gram-negative bacteria. Storage solutions C and E reduced the abundance of Bifidobacterium, whereas storage solution D increased; except that preservation solution E relatively reduced the abundance of Lactobacillus, but the preservation solution A, B, C, and D were all closer to the control. Except for the greater difference in preservation solution D, preservation solution C was the closest to the control group on Streptococcus; preservation solution D reduced Ruminococcaceae UCG 003 than the control group. However, other preservation solutions were not much different from the control group; different preservation solutions increased the abundance of Escherichia-Shigella than the control group, and preservation solutions A and B increased the abundance of Klebsiella, but preservation solution C, D, and E were closer to the control group. Overall, preservation solution C performed better in stabilizing the composition of the gut microbiota. This study provides reference for standardized microbiome projects. Subsequent research can choose a targeted preservation solution and preservation time based on this study.