This study is to investigate the antitumor activity of ophiopogonin B (OP-B).  MTT assay, flow cytometric analysis, acridine orange staining, Lyso-Tracker Red staining and HeLa-GFP-LC3 transfect cells assay were used to detect the proliferation activity, apoptosis and autophagy of HeLa cells.  The results showed that OP-B exerted potent antiproliferative activity on HeLa cells, the cell growth inhibition effect of OP-B was not due to apoptosis and OP-B could induce autophagy of HeLa cells.  OP-B also induced the protein expression up-regulation of Beclin-1 and promoted LC3 I transformation LC3 II, which were representative proteins of autophagy.  Furthermore, 3-MA, an inhibitor of autophagy, not only inhibited OP-B-mediated autophagy but also almost completely reversed the antiproliferative effect of OP-B, suggesting that the growth inhibition effect of OP-B was autophagy dependent.  Western blotting demonstrated that OP-B inhibited the phosphorylation of Akt and its’ downstream vital protein, such as mTOR and p70S6K. In addition, OP-B also induced the protein expression up-regulation of PTEN, which is a negative regulation protein for Akt/mTOR signaling pathway.  However, OP-B did not affect the protein expression of total Akt.  Collectively, the antitumor effects of OP-B were autophagy-dependent via repression Akt/mTOR signaling pathway.  Therefore, OP-B is a prospective inhibitor of Akt/mTOR and may be used as an alternative compound to treat cervical carcinoma.