In vitro experiments were conducted to study the effects of Onchidium reevesii polysaccharides and sulfated Onchidium reevesii polysaccharides on the growth of Hela human cervical cancer cells, and with positive drug cisplatin (0.5, 0.75, 1.5 μg/mL) as control. Different mass concentrations (50, 75, 150 μg/mL) of OSP and S-OSP were applied to Hela cells, and we used CCK-8 method to analyze the proliferation of Hela cells at 12, 24, 48 and 72 h, and analyzed the cell cycle with flow cytometry, and used qRT-PCR to detect the expression changes of related apoptosis genes. The results show that both OSP and S-OSP can reduce the viability of Hela cells and induce their apoptosis, moreover, S-OSP can more significantly inhibit the proliferation activity of Hela cells than OSP, It shows that the sulfation modification changes the structure of the polysaccharide and affects the anti-tumor activity of the polysaccharide. After 48 hours of treatment, the growth inhibition rates of Hela cells in the three concentrations of OSP group were 60%, 67%, and 71%, respectively; the growth inhibition rates of Hela cells in the S-OSP group were 74%, 77%, and 84%, respectively; the growth inhibition rates of Hela cells in the cisplatin group were 82%, 86%, and 90%, respectively. Flow cytometry showed that after OSP and S-OSP had acted for 24 h, the ratio of early and late apoptosis of cells was significant, and increased proportion of cells in G0/G1 phase, and the proportion of S-phase cells continued to decrease; Real-time fluorescence quantification showed that the expression of pro-apoptotic gene Caspase-3 increased significantly with the increase of drug mass concentration, and the expression of anti-apoptotic genes Bcl-2 and Bcl-xL did not change significantly with the increase of drug mass concentration. It shows that OSP and S-OSP can induce Hela cell apoptosis by regulating the Caspase signaling pathway.