The contamination of pesticides has been recognized as a major stressor in fresh water ecosystems in terms of the losses of services and population declines and extinctions. However, information on the adverse effects of pesticides on zooplankton communities under natural field conditions are still lacking, although zooplankton is quite sensitive to most of pesticides in laboratory studies. In this study, a natural lake ecosystem (Liangzi Lake) was used to determine the relationship between pesticide contamination and abundance decline of metazoan zooplankton. In August 2020, the comprehensive trophic level indexes and the abundance of phytoplankton in the 14 sampling sites of Liangzi Lake were comparable, but the abundance of metazoan zooplankton showed significant variations across two orders of magnitude. These results suggested that other factors, such as pesticide contamination, might be responsible for the variations of metazoan zooplankton community. Furthermore, the responsible pesticides were screened, and totally 29 pesticides were obtained. Finally, five pesticides were identified to provide more than 99.4% toxic contributions and chlorpyrifos and cypermethrin were two main causal agents. These results were further supported by laboratory exposure experiments using D. magna and field study in November 2020, where the concentrations of the 29 pesticides were strongly decreased and the abundance of metazoan zooplankton was comparable across the 14 sites of Liangzi Lake. Taken together, this work provided an evidence that the contamination of pesticides might be responsible for the abundance decline of metazoan zooplankton in a natural freshwater ecosystem.