The long suspicion of the potential harm of carbon dioxide (CO2) pneumoperitoneum exists in laparoscopic cancer surgery. For better understanding of this problem, we targeted this study at the effects of CO2 pneumoperitoneum on the invasive ability of ovarian carcinoma cell line and the possible mechanism within it. To study the effects of CO2 pneumoperitoneum on carcinoma cell, SKOV3 cells were divided into 2 groups, respectively exposed to pneumoperitoneal CO2-insufflation and normal conditions. To study the possible mechanism, SKOV3 cells were divided into 3 groups, one was exposed to CO2 pneumoperitoneum, one to N2 and the other to normal conditions served as control. The in vitro adhesive and invasive ability of the cells was analyzed through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and Boyden filters metastasis model; the expressions of vascular endothelial growth factor C (VEGF-C) and matrix metalloproteinase 9 (MMP9) were determined by reverse transcription polymerase chain reaction( RT-PCR), and Western blot. We found that the adhesive ratio of SKOV3 cells exposed to CO2 was significantly higher than that of the control group; cells exposed to CO2 invaded the matrigel with a greater number (P＜0.01); the expression of VEGF-C exposed to both CO2 and N2 was significantly increased compared with control group (P＜0.05); the MMP9 expression level of CO2 group was higher than that of N2 group, P＜0.05. We concluded that carbon dioxide pneumoperitoneum may improve the adhesive and invasive ability of ovarian carcinoma cell line in vitro and CO2 can also be an independent factor to stimulate the expression of MMP9.