Matrix metalloproteinases (MMP), tissue inhibitors of metalloproteinases (TIMP), and MMP-TIMP interactions may contribute to the highly programmed process of embryo implantation. The loss of the delicate MMP-TIMP balance may lead to abnormal implantation. The role of TIMP-4 in mouse implantation has not been reported. This study examined mRNA and protein expression levels of TIMP-4 in the blastocyst and uteri of pregnant mice. We also investigated the effects of a specific TIMP-4 antibody on embryo outgrowth and on the gene and protein expression levels of two gelatinases. High levels of TIMP-4 mRNA and protein were detected in day 3-5 embryos and in the trophoblast cells of mice blastocysts, suggesting that TIMP-4 may be involved in embryo implantation. Furthermore, TIMP-4 antibody promoted blastocyst outgrowth in a dose-dependent manner, but had no effect on blastocyst adhesion to extracellular matrix. A specific TIMP-4 antibody also increased mRNA and protein expression levels and the enzymatic activities of gelatinase A (MMP-2) and gelatinase B (MMP-9). This study suggests that TIMP-4 may restrict mouse blastocyst outgrowth and embryo implantation by inhibiting the activities of MMP-2 and -9.