The objective of this study was to determine the effects of imazethapyr on soil microbial communities combined with its effect on soybean growth. A short-term field experiment was conducted, and imazethapyr was applied to the soil at three different doses [1-fold, 10-fold, and 50-fold of the recommended field rate (H1, H10, H50)] during the soybean seedling period (with two leaves). Soil sampling was performed after 1, 7, 30, 60, 90, and 120 days of application to determine the imazethapyr concentration and microbial community structure by investigating phospholipid fatty acids (PLFA) and microbial biomass carbon (MBC). The half-lives of the imazethapyr in the field soil varied from 30.1 to 43.3 days. Imazethapyr at H1 was innocuous to soybean plants, but imazethapyr at H10 and H50 led to a significant inhibition in soybean plant height and leaf number. The soil MBC, total PLFA, and bacterial PLFA were decreased by the application of imazethapyr during the initial period and could recover by the end of the experiment. The ratio of Gram-negative/Gram-positive (GN/GP) bacteria during the three treatments went through increases and decreases, and then recovered at the end of the experiment. The fungal PLFA of all three treatments increased during the initial period and then declined, and only the fungal PLFA at H50 recovered by the end of the treatment. A principal component analysis (PCA) of the PLFA clearly separated the treatments and sampling times, and the results demonstrate that imazethapyr alters the microbial community structure. This is the first systemic study reporting the effects of imazethapyr on the soil microbial community structure under soybean field conditions.