Paeoniflorin is one of the active ingredients of the commonly used herbal medicine derived from Paeonia, which exhibits anticancer properties. MicroRNA-16 (miR-16) is upregulated in CD133(-) cells, but downregulated in CD133(+) cells from glioma tissue. Matrix metalloproteinase-9 (MMP-9) expression in glioma tissue samples is significantly higher than that in healthy brain tissue samples. Therefore, miR-16 and MMP-9 expression may be associated with glioma pathogenesis. In the present study, the effects of paeoniflorin on glioma were analyzed. U87 cells were treated with paeoniflorin at 0, 5, 10 and 20 μΜ concentrations. The results suggested that paeoniflorin inhibited U87 cell proliferation and accelerated cell apoptosis. In the present study paeoniflorin treatment increased miR-16 expression and reduced MMP-9 protein expression in U87 cells. Additionally, the results of the present study suggested that miR-16 may regulate MMP-9 expression in miR-16-transfected U87 cells. Furthermore, anti-miR-16 antibodies were used in order to investigate the apoptotic effects of paeoniflorin on U87 cells. The results demonstrated that paeoniflorin inhibits proliferation and induces apoptosis of human glial cells, via miR-16 upregulation and MMP-9 downregulation.