Objective To investigate the influence of different pressures and duration of CO2 pneumoperitoneum on the adhesive and invasive ability of gastric cancer cells based on the expressions of adhesive and invasive molecules. Methods With an artificial CO2 pneumoperitoneum model in vitro, human gastric cancer cell lines including MKN-45, SGC-7901, and MKN-28 were exposed to CO2 in different environments: 0 mm Hg （1 mm Hg=0.133 kPa）, 9 mm Hg （2 h, 4 h）, and 15 mm Hg （2 h, 4 h）. The expressions of mRNA of E-cadherin, intercellular adhesion molecule-1 (ICAM-1), matrix metalloproteinase-2 (MMP-2), and vascular endothelial growth factor-A (VEGF-A) in the different environments were measured by RT-PCR. The expressions of protein of E-cadherin and ICAM-1 in the environments of 0 mm Hg and 15 mm Hg （4 h） were measured by FCM. Results With the increase of duration or pressure, RT-PCR showed that there was a downward trend in the expression of E-cadherin mRNA as well as there were upward trends in the expressions of ICAM-1, MMP-2, and VEGF-A mRNA; FCM showed that there was a downward trend in the expression of E-cadherin protein while the expression of ICAM-1 protein showed the opposite change. But there were no obvious differences under different environment (P＞0.05). Conclusions Under low pressure (≤15 mm Hg) and short time (≤4 h) of CO2 pneumoperitoneum, the adhesive and invasive ability of gastric cancer cells could not be affected, which means that under this environment, CO2 pneumoperitoneum will not increase the possibility of neoplasm metastasis.