Neonicotinoid pesticides are the most widely used insecticides worldwide and have become a global environmental issue. Previous studies have shown that imidacloprid, the most used neonicotinoid, can negatively affect a wide range of organisms, including non-target insects, fish, invertebrates, and mammals. Imidacloprid can also accumulate and persist in soils, posing threats to the terrestrial ecosystem. However, we know little about one ecologically important group of organisms, the single-celled soil protists. In this study, we used a soil amoeba, Dictyostelium discoideum, to test whether and how imidacloprid affects the growth and development of soil amoebae. We provide the first empirical evidence that environmental concentrations of imidacloprid negatively impact the fitness and development of soil amoebae. In addition, the adverse effects did not show a dose-response relationship with increased imidacloprid concentrations, where no significant difference was observed among the treatment groups. Further transcriptome analyses showed that imidacloprid affected amoeba's key DEGs related to phagocytosis, cell division, morphogenesis, and cytochrome P450. Moreover, soil amoebae show both conserved and novel transcriptional responses to imidacloprid. In conclusion, this study has expanded the non-target list of imidacloprid from animals and plants to single-celled protists, and we believe the impact of neonicotinoid pesticides on the microbiome is significantly underestimated and deserves more studies.