To understand the response of murine uterine natural killer (uNK) cells to Toll-like receptor (TLR) 3 agonist at the early gestation stage, CBA×DBA/2 mice were intraperitoneally (i.p.) injected with polyinosinic–polycytidylic acid (poly I:C), the specific TLR3 agonist, at a dose of 10 μg/g BW or PBS at gestation day (gd) 6.5. The CD69 expression of uNK (DX5 + CD3 − ) cells was highly up-regulated and reached 92.3±0.9%, the percentage of intracellular TNF-α + or IFN-γ + uNK (DX5 + CD3 − ) cells in the implantation sites of CBA×DBA/2 matings was also significantly increased 24 h after poly I:C injection. Surprisingly, poly I:C treatment significantly decreased the total number of uNK cells (either DX5 + CD3 − or DBA + ) at fetal–maternal surface, but had no influence on local NKT cells, T cells and DCs. This investigation will help to explain the central role for hyperactivated uNK cells in the progress of mice pregnancy.