Objective To investigate the impact of levonorgestrel (LNG) on endometrial stem cell (EMSC) activity via the estrogen-Notch signaling axis. Methods 60 rats wevedivided intocontial group with sham surgery (10 rats) and model of endometriosis (50 rats). The modeled rats were then randomly divided into five groups: untreated model group, LNG group receiving LNG injections,LNG+ER group given LNG and transfected with ER-α cDNA, an LNG+ER+siNC group transfected with LNG, ER-α cDNA, and siRNA NC plasmid, and an LNG+ER+siNotch1 group transfected with LNG, ER-α cDNA, and Notch1 siRNA plasmid. Parallel in vitro experiments were conducted using EMSCs isolated from both sham surgery and endometriosis model mice, which were cultured and divided into similar treatment groups, all with added estrogen. Sham surgery-derived EMSCs served as an untreated blank control. Observations focused on the interplay between estrogen and the ER-Notch signaling pathway. Cultured EMSCs from ectopic endometrium were further grouped into four: control group, estradiol (E2) group receiving exogenous 17β-estradiol, E2+ICI group treated with estrogen receptor inhibitor ICI plus E2, and Notch inhibition group treated with the Notch signal blocker DAPT along with E2. Pathological changes, vascular density, and EMSC proliferation, migration, differentiation, as well as estrogen, ER, Notch1, Hes1, and Hey2 expression levels, were assessed.Results Indicated that compared to the sham surgery group, the model group showed significantly elevated levels of estrogen, ER-α, Notch1, Hes1, Hey2, vascular density, EMSC counts, proliferation, migration, and differentiation rates (P<0.05). These parameters were significantly reduced in the LNG group compared to the model group (P<0.05). When compared to the LNG group, the LNG+ER group exhibited significant increases in ER-α, Notch1, Hes1, Hey2, vascular density, EMSC activity, and proliferation, migration, and differentiation rates (P<0.05). However, these increases were reversed in the LNG+ER+siNotch1 group, showing significantly lower levels than the LNG+ER group (P<0.05), with no significant difference observed between the LNG+ER and LNG+ER+NC groups (P>0.05). Supplementation with E2 led to significant increments in estrogen, ER-α, Notch1 expression, and cellular activities. Conversely, application of the ER inhibitor ICI or the Notch inhibitor DAPT alongside E2 resulted in significant decreases in ER and Notch1 expression and cellular proliferation, migration, and differentiation rates (P<0.05).Conclusion LNG can reduce the activity of endometrial stem cells by down regulating estrogen Notch signal axis, so as to inhibit the development of endometriosis