Catalpol is expected to possess diverse pharmacological actions including anti-cancer, anti-inflammatory and hypoglycemic properties. Matrix metalloproteinase-2 (MMP-2) is closely related to the pathogenesis of ovarian cancer. In addition, microRNA-200 (miR-200) can modulate phenotype, proliferation, infiltration and transfer of various tumors. Here, OVCAR-3 cells were employed to investigate whether the effect of catalpol (25, 50 and 100 μg/mL) promoted apoptosis of ovarian cancer cells and to explore the potential mechanisms. Our results demonstrate that catalpol could remarkably reduce the proliferation and accelerate the apoptosis of OVCAR-3 cells. Interestingly, our findings show that catalpol treatment significantly decreased the MMP-2 protein level and increased the miR-200 expression level in OVCAR-3 cells. Further, microRNA-200 was shown to regulate the protein expression of MMP-2 in OVCAR-3 cells. It is concluded that catalpol suppressed cellular proliferation and accelerated apoptosis in OVCAR-3 ovarian cancer cells via promoting microRNA-200 expression levels and restraining MMP-2 signaling.