There is accumulating evidence that cancer stem cells (CSCs) play an important role in tumor progression. Novel strategies targeting CSCs have been widely researched. In the present study, we explored whether such CSCs existed in human ovarian cancer (OVCA) cell line and whether anti-CD44 antibody had effects on such subpopulation. We isolated and identified spheroid cells from SKOV-3. Then we used A3D8, an anti-CD44 mAb to treat spheroid cells with so-called “stemness”. Effects of A3D8 on spheroid cells’ biological behaviors were examined. Our findings showed that there was a small subpopulation that had so-called “stemness” in SKOV-3 cell line. Against spheroid cells, A3D8 can (1) inhibit cell proliferation; (2) change cell cycle distribution and expression of p21, CDK2 and cyclinA; (3) enhance cisplatin (DDP)-induced apoptosis; (4) promote cell differentiation; (5) inhibit clone formation efficiency; (6) reduce invasive efficacy; (7) inhibit tumorigenicity. Thus, to sum up points which we have just showed, spheroid cells isolated from SKOV-3 can be used as an appropriate in vitro model for relevant study of human ovarian CSCs. And our results reasoned that anti-CD44 therapy may become a potential promising strategy for OVCA treatment. There are accumulating evidence that cancer stem cells (CSCs) play an important role in tumor progression. Novel strategies targeting CSCs have been been researched in tumor. In the present study, we explored whether such CSCs existed in human ovarian cancer (OVCA) cell line and whether Anti-CD44 antibody had effects on such subpopulation. We isolated and identified spheroid cells from SKOV-3. Then we used A3D8, an anti-CD44 mAb to treat spheroid cells with so-called “stemness”. Effects of A3D8 on spheroid Our findings showed that there was a small subpopulation that had so-called “stemness” in SKOV-3 cell line. Against spheroid cells, A3D8 can (1) inhibit cell proliferation; (2) change (3) enhance cisplatin (DDP) -induced apoptosis; (4) promote cell differentiation; (5) inhibit clone formation efficiency; (6) reduce invasive efficacy; (7) inhibit Thus, to sum up point s which we have just showed, spheroid cells isolated from SKOV-3 can be used as an appropriate in vitro model for relevant study of human ovarian CSCs. And our results reasoned that anti-CD44 therapy may become a potential promising strategy for OVCA treatment.