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.