Abstract: Objective To explore the effect of melatonin on endometrial stromal cells (ESC) in endometriosis and its possible mechanism. Methods The eutopic endometrial tissues of 13 patients with endometriosis were collected for primary isolation and mesenchymal cells were cultivated. The cells were treated with 0.25, 0.5 and 1 mmol/L melatonin respectively, before detection of cell viability by CCK-8 assay. Transwell assay was used to detect cell migration and invasion. qRT-PCR was used to detect the expression of fibrinogen α chain (FGA) mRNA. Western blot was used to detect the expression of keratin, vimentin, slug, FGA, p-FAK, FAK, p-AKT, AKT and MMP-2 protein. Results ESC cells were successfully isolated and cultured. Compared with the blank control group, treatment of ESC cells with 0.25 mmol/L, 0.5 mmol/L, and 1 mmol/L melatonin resulted in significant decreases in the cell viability, cell migration and invasion numbers (P＜0.05), and increases in the expression of keratin protein (P<0.05), and decreases in the expression of vimentin, slug, FGA, p-FAK/FAK, p-AKT/AKT and MMP-2 protein (P<0.05). The effect of melatonin on ESC cells was dose-dependent. Compared with the melatonin +oe-NC group, the melatonin+oe-FGA group presented significant increases in cell viability, cell migration and invasion number (P<0.05), and decreases in the expression of keratin protein (P<0.05), and increases in the expression of FGA mRNA and protein, the expression of vimentin, and the levels of slug, FGA, p-FAK/FAK, p-AKT/AKT and MMP-2 (P<0.05). Conclusions Melatonin may inhibit ESC cell migration, invasion and epithelial-mesenchymal transition by inhibiting FGA-mediated FAK/AKT/MMP-2 pathway activation, thereby inhibiting the progression of endometriosis.