Abstract: Objective To determine the expression of microRNA-221 (miR-221) in endometrial tissues and its impact on the proliferation of ectopic endometrial stromal cells. Methods Endometrial stromal cells were isolated, cultured and identified from normal endometrial tissues (taken from patients without endometriosis) and ectopic endometrial tissues (taken from patients with ovarian endometriosis). The expression of microRNA-221 was detected by stem-loop qRT-PCR. Changes in the expression of miR-221-3p in endometrial stromal cells exposed to estraldiol (10^-8 mol/L) for 48 h were detected. The effects of miR-221-3p inhibitor on the expressions of miR-221-3p, phosphatase and tensin homology deleted on chromosome ten (PTEN) and cell proliferations were compared with those of the negative control (NC, 10 nmol/L). Results The expression of miR-221-3p in ectopic endometrial tissues was 4.2 times higher than that in normal endometrial tissues (P=0.039):2.66 times higher in ectopic endometrial stromal cells compared with normal endometrial stromal cells (P=0.029). But no differences in the expression of miR-221-5p were found (P>0.05). No differences in the change of miR-221-3p expression after exposure to estrogen for 48h were found between normal and ectopic stromal cells. Inhibition of miR-221-3p function was associated with decreased cell proliferation (P=0.018) and increased expression of PTEN gene (P=0.021). Conclusion The expression of microRNA-221 is upregulated in ectopic endometrial tissues and ectopic endometrial stroma cells. Inhibiting the function of miR-221-3p may result in increased PTEN expression and decreased cell proliferation in endometrial stromal cells.