One of the widely used techniques in Drosophila is the Gal4/UAS system, which allows tissue-specific misexpression or knockdown of interested genes. The original UAS vector, the pUASt, can only be activated in somatic tissues, not in the germline. By replacing the hsp70 promoter and the SV40 3’UTR with the P transposase promoter and the K10 3’UTR, respectively, Rørth (1998) generated a modified UAS vector, called pUASp, which can respond to Gal4 in both somatic and germline tissues. However, the underlying mechanisms for UASt silencing in germline cells remained unclear. Here, we report that the piRNA pathway is involved in suppressing UASt expression in ovarian germline cells. When piRNA pathway components (Piwi, AGO3, Aub, Spn-E, and Vasa) were knocked down individually in germline cells, the UASt-RFP/GFP can be detected in germline cells in the ovary. To determine how piRNAs silence UASt-transgene expression, we performed RNA-seq analyses of small RNAs and found that the hsp70 promoter of pUASt is a potential piRNA target. Additionally, since the UASt vector also contains the SV40 3’UTR, which happens to be targeted by the Nonsense-mediated RNA decay (NMD) pathway. To find out whether NMD is also responsible for UAST silencing in the germline, we knocked down several NMD key components in ovarian germline cells, and observed a low frequency of UASt-RFP expression in germline cells with Smg5 knockdown. Taken together, our findings suggest an important role of the piRNA pathway, and a potentially minor role of the NMD pathway in germline UASt silencing.