We constructed shRNA vectors with different stem length, and tested the silencing effectiveness in mouse cells and embryos. We designed interfering RNAs with stems of 21 bp, 27 bp, and 29 bp. The enhanced green fluorescent protein gene was used as target gene. The synthesized single strands were annealed and cloned into psiSTRIKE and the recombinant plasmids (EGFP-21 siRNA, EGFP-27 siRNA, and EGFP-29 siRNA) were transfected into the mouse embryonic fibroblast with lipofection. The mRNA expression level of the enhanced green fluorescent protein gene was checked by real-time quantitative PCR. The silencing effectiveness of the 29 bp shRNA vector was stronger than which of the 21 bp and 27 bp. The findings in this study are of interest for selecting the hairpins for mouse individuals.