Lanosterol 14α-demethylase (LDM) is expressed ubiquitously in all mammals and is important in cholesterol biosynthesis. However, whether LDM expression is involved in the interaction between uterus and embryo during implantation remains unknown. In the present study, the expression of LDM was investigated in mouse embryo and uterus during the peri-implantation period using confocal microscopy, immunohistochemistry and western blot methods. Further, regulation of LDM expression was investigated in pseudopregnancy, delayed implantation, artificial decidualisation and ovariectomisation using 17β-oestradiol and progesterone treatment mouse models. The results showed that LDM was selectively expressed in preimplantation embryos and the uterine subluminal stroma surrounding the implanting blastocyst on Day 5 of pregnancy. No corresponding signal was detected in the uterus on Day 5 of pseudopregnancy. Most notably, once delayed implantation was terminated by oestrogen treatment and the embryo implanted, a high level of LDM expression was induced in the subluminal stroma surrounding the implanting blastocyst, whereas no corresponding signal was detected in the delayed implantation uterus. A high level of LDM expression was observed in the uterus decidua on Days 6–8 of pregnancy. Furthermore, LDM expression was induced in the uterine stroma under artificial decidualisation. Oestrogen, but not progesterone, treatment induced a high level of LDM expression in the uterus of ovariectomised mice. These results indicate that LDM is closely related to mouse embryo implantation and can be upregulated by oestrogen.