Small RNAs derived from repetitive sequences appear to play essential roles in mammalian gametogenesis and early development. In this study we focused on the short interspersed nucleotide element B1 (SINE/B1) small RNAs, which were zygotically expressed in pre-implantation mouse embryos; and we investigated whether the SINE/B1 small RNAs played an active role in gene silencing during early mouse development. The results indicated that the level of silencing activity involving the SINE/B1 small RNAs as mediators was significantly reduced in Dicer-knockdown mouse embryos. In addition, when the SINE/B1 small RNAs were mapped to a full-length SINE/B1 sequence, phase-distribution of the small RNAs appeared, suggesting possible enzymatic involvement. Therefore, our present study suggested that the zygotically expressed SINE/B1 small RNAs in pre-implantation mouse embryos contain active small RNAs, which were presumably processed by Dicer and involved in gene silencing.