The Argonaute/PIWI protein family consists of Argonaute and PIWI subfamilies. Argonautes function in RNA interference and micro-RNA pathways; whereas PIWIs bind to PIWI-interacting RNAs and regulate germ line development, stem cell maintenance, epigenetic regulation, and transposition. However, the role of PIWIs in mammalian stem cells has not been demonstrated, and molecular mechanisms mediated by PIWIs remain elusive. Here we show that MILI, a murine PIWI protein, is expressed in the cytoplasm of testicular germ line stem cells, spermatogonia, and early spermatocytes, where it is enriched in chromatoid bodies. MILI is essential for the self-renewing division and differentiation of germ line stem cells but does not affect initial establishment of the germ line stem cell population at 7 days postpartum. Furthermore, MILI forms a stable RNA-independent complex with eIF3a and associates with the eIF4E- and eIF4G-containing m7G cap-binding complex. In isolated 7 days postpartum seminiferous tubules containing mostly germ line stem cells, the mili mutation has no effect on the cellular mRNA level yet significantly reduces the rate of protein synthesis. These observations indicate that MILI may positively regulate translation and that such regulation is required for germ line stem cell self-renewal.