Abstract: Objective To observe the effect of CO₂ on the anti-anoikis of HeLa cell (cervical cancer). Methods HeLa cells were randomly divided into experimental group and control group. In experimental group, CO₂ was insufflated into the containers by pressures of 8 mm Hg for 4 hours. Control group was not treated by CO₂. Two groups of cells were cultured for 24h, 72h and 120h in adherent and suspension methods. The effect of CO₂ on the proliferation and antianoikis of HeLa cells were detected through soft agar colony formation experiments, Poly HEMA suspension culture and Flow cytometry assay. Results The abilities of apoptosis and anti-anoikis were observed in 2 groups. The proliferation and colony formation capabilities were stronger in experimental group than that in control group, but the anti-anoikis of experiment group was significantly lower than that of the control group(P＜0.01). Conclusion CO₂ was able to improve the colony formation and anoikis of HeLa cells, no a great impact, however, on anti-anoikis.