The anti-implantation effects of high oestradiol (E2) concentrations could be mediated by E2 metabolites. Herein, we examined whether 2-methoxyoestradiol (2ME) impairs embryo implantation via its target protein F-spondin. Mice on Day 3 of pregnancy were treated with E2 concomitantly with the cathecol-O-methyl transferase inhibitor OR486 and the number of implanted embryos was recorded 5 days later. The effect of 2ME or 4-methoxyoestradiol (4ME) on embryo implantation was also investigated. Plasma and uterine levels of 2ME were measured 0.5, 1 or 3h after E2 treatment while the mRNA for spondin 1 (Spon1) and F-spondin were determined in the uterus 3, 6, 12 or 24h after 2ME treatment. Finally, the effect of a neutralising F-spondin antibody on the anti-implantation effect of 2ME was explored. OR486 blocked the anti-implantation effect of E2; 2ME, but not 4ME, affected embryo implantation. The 2ME concentration was increased after 0.5 and 1h in plasma and 3h in uterine fluid following E2 treatment. 2ME increased levels of Spon1 at 12 and 24h although F-spondin was increased at 12h. F-spondin antibody blocked the effect of 2ME on embryo implantation. We conclude that 2ME impairs mouse embryo implantation via activation of F-spondin in the uterus.