PIWI family members miwi and mili as a constituent part of the piRNA pathway play a crucial role in germline maintenance, differentiation and meiosis conserved in mammals. A previous study shows estrogen could regulate the piwi pathway and determine the gonadal sex differentiation in lower animals. In this study, we expose mouse testis relative cells or tissues in different concentrations and a continuous time series, using quantitative RT-PCR, flow cytometry and immunohistochemical techniques for analyses of the influence over miwi and mili in vitro and in vivo. The in vitro studies show estrogen can downregulate the expression of miwi and mili. A similar pattern is observed in the first several days and then later back to normal in vivo. Our result indicates that estrogen can decrease expression of miwi and mili in male testis.