Glyphosate (GLP) is a globally ubiquitous herbicide that poses a threat to living organisms due to its widespread presence in soil ecosystems. However, the results of current research regarding the effects of glyphosate on soil microorganisms and its ecological risks are vague and inconsistent. In this study, we investigated the impact of single (low/high-dose) and reapplication (high-dose) of glyphosate applications on soil microbes through indoor incubation experiments using 16S rRNA gene high-throughput sequencing technology. Our findings indicate that in the short term, whether it's single or reapplication glyphosate applications, changes in diversities of soil bacterial community were less than those in community composition. Glyphosate exerts selective pressure on soil microbial communities, resulting in a predominant process of species replacement after glyphosate application, and quantitative analysis revealed a higher turnover rate of microbial communities under glyphosate reapplication. Factors related to nitrogen cycling, especially NH