Gene knockdown using double-stranded RNA (dsRNA) is a powerful tool to characterize gene function in Drosophila . The Drosophila germline provides an elegant model to study the regulation of numerous processes, such as stem cell fate, gametogenesis, piRNA, and piRNA-related gene functions. However, for unknown reasons, traditional dsRNA gene knockdown has not been successful in the germline. Here, we establish a simple gene silencing method for the Drosophila germline by the generation of artificial miRNA-mimicking siRNAs. This method, which is different from that of classical dsRNA, mimics natural miRNA biogenesis and enables the analysis of gene functions specifically in the Drosophila germline.