The endometrial-myometrial interface (EMI) is an important region of the human uterus, which has attracted little research attention. This mucosal-muscular interface has characteristic features when compared with other similar interfaces in the human body. It lacks an intervening tissue layer and as a result, the endometrium sits directly on the myometrium rendering it vulnerable to invasion by the endometrium. Both endometrium and myometrium are sensitive to sex steroids, and their structure and function depend to a large extent on the sex hormonal milieu. Endometrium, which forms one border of the EMI, is a complex tissue consisting of several polarized microenvironments. At a cellular level, sex steroids interact with local mediators secreted by a variety of cell types and are important in maintaining the complex structure and function of the endometrium. Basal endometrium contains prominent aggregates of leukocytes that may be important in controlling local cell growth and function. Myometrium also has a distinct zonal anatomy. The recently described junctional zone differs structurally and functionally from the outer myometrium, although these functions are not yet clearly understood. Embryologically, it originates from müllerian ducts together with endometrium, whereas the outer myometrium has a non-müllerian origin. During early pregnancy, the EMI is disturbed by invading trophoblast. Alterations of myometrial intercellular matrix proteins together with expression of appropriate receptors by the trophoblast seem to regulate this unique interaction. The EMI also is disrupted in adenomyosis. The sequence of events taking place at the EMI during development of this pathology is still debated.