One purified polysaccharide protein tyrosine phosphatase (PTP) was isolated from the roots of Polygala tenuifolia. The aim of the present study is to investigate the antitumor effect of PTP on human ovarian cancer OVCAR-3 cells and explore the molecular mechanism of the action involved. The results of MTT assay and apoptosis detection assay showed that PTP inhibited cellular proliferation of OVCAR-3 cells and induced apoptotic cellular death via arresting cell circle at the G0/G1 phase. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis identified that bcl-2 gradually decreased at both transcription and protein levels after PTP treatment for 48 h in OVCAR-3 cells, while those of bax, cytochrome c, caspase-3, and caspase-9 increased. In addition, the low expression of NF-κB in PTP-treated OVCAR-3 cells would trigger the extrinsic pathway of programmed cell death signaling in tumor cells. These results together suggest that PTP may induce apoptosis of OVCAR-3 cells through a mitochondrial pathway.