Objective To investigate the effect of targeted interruption of cyclooxygenase-2 (COX-2) gene by small interference RNA (siRNA) on the expression of COX-2, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) in eutopic and ectopic endometrial stromal cells (ESC) with endometriosis, and the effect on the content of 6-keto-prostaglandin-F1α (6-keto-PGF1α, metabolites of COX) and the apoptosis of eutopic and ectopic ESC with endometriosis. Methods Ectopic and eutopic ESC from 30 women with endometriosis were isolated and cultured respectively. Then, ESC were classified into three groups: interference group, negative control group and blank control group. ESC in interference group were injected into siRNA transfection complex while ESC in negative control group were injected into negative control transfection complex. ESC from 10 participants without endometriosis were the normal control group. The mRNA and protein expression of COX-2, VEGF, MMP-9 in pre-transfected and post-transfected eutopic and ectopic ESC were detected through real time reverse transcription PCR and western blot. The content of 6-keto-PGF1α was determined by ELISA, the apoptotic cells were detected by flow cytometry. Results After interruption of COX-2 gene, there were no significant difference in the mRNA and protein expression of COX-2, VEGF and MMP-9 between the negative control group and blank control group (P>0.05); the mRNA and protein expression of the three genes in interference group were significantly lower than those in negative control group and blank control group (P<0.05); the mRNA expression of the three genes in interference group of eutopic ESC were 0.87±0.06, 1.76±0.59, 1.04±0.32, in interference group of ectopic ESC were 0.75±0.12, 1.62±0.47, 0.88±0.25, the protein expression of the three genes in interference group of eutopic ESC were 0.457 ± 0.019, 0.500 ± 0.012, 0.361 ± 0.008, in interference group of ectopic ESC were 0.323 ± 0.018, 0.474 ± 0.016, 0.339 ± 0.009;the mRNA and protein expression of the three genes in ectopic ESC had a more reduction than those in eutopic ESC (P<0.05). The results from ELISA revealed that the content of 6-keto-PGF1α in the normal control group [(17.7 ± 1.9) pg/ml] were significantly lower than those in the blank control group (P<0.05), the content of 6-keto-PGF1α in ectopic ESC were significantly higher than that in eutopic ESC (P<0.05), the content of 6-keto-PGF1α in the blank control group of eutopic and ectopic ESC were (32.4±2.6) pg/ml, (38.2±3.7) pg/ml;there was no significant difference in the content of 6-keto-PGF1α between the negative control group and blank control group (P>0.05);compared with those of negative control group and blank control group, the content of 6-keto-PGF1αin interference group decreased significantly (P<0.05), the content of 6-keto-PGF1α in interference group of eutopic and ectopic ESC were (17.1 ± 2.4) pg/ml, (20.9 ± 2.7) pg/ml; the content of 6-keto-PGF1α in eutopic ESC had a slightly more reduction than that in ectopic ESC (P>0.05). The results from flow cytometry displayed that, there was no significant difference in apoptotic cells between the negative control group and blank control group (P>0.05);compared with those of negative control group and blank control group, more apoptotic cells were detected in interference group and the difference was significant (P<0.01);the apoptotic cells in ectopic ESC were significantly more than that in eutopic ESC (P<0.05); the apoptosis rate in interference group of eutopic and ectopic ESC were (33.76 ± 0.06)%, (47.18 ± 0.12)%. Conclusions Our results suggested the targeted interruption of COX-2 gene by siRNA effectively inhibited the mRNA and protein expression of COX-2, VEGF and MMP-9 in both eutopic ESC and ectopic ESC with endometriosis, greatly increased the apoptotic rate of cells and obviously reduced the content of 6-keto-PGF1αby inhibiting the activity of COX-2. And the changes in ectopic endometrium were more evident than those in eutopic endometrium.