Objective To investigate the effect of inhibiting mitochondrial respiratory chain complex Ⅰ on the migration and invasion capacity of colon cancer cell line Caco2, and to explore the possible molecular mechanism. Methods Human colon cancer cell line Caco2 was treated with 1 μmol/L rotenone in vitro. Then the relative activity of mitochondrial respiratory chain complex Ⅰ was examined by chromatometry, the capacity of cell migration and invasion was determined by transwell assay, and the reactive oxygen species (ROS) level in cells was determined using flow cytometry. Results The activity of mitochondrial respiratory chain complex Ⅰ of Caco2 cells treated with 1 μmol/L rotenone was significantly lower than that of the untreated cells(P<0.01). In addition, Transwell assay showed that the cell migration rate and invasive rate in Caco2 cells treated with rotenone were significantly higher than those in untreated Caco2 cells after 48 h (migrant rate [30.4±1.4]% vs [22.6±1.4]%, invasive rate [20.3±1.0]% vs [15.2±1.3]%, P<0.01). Furthermore, the ROS level in the rotenone treated cells was significantly higher than that in untreated cells ([5.68±0.44]% vs [3.46±0.30]%, P<0.01). Conclusion Our data suggest that inhibiting the activity of mitochondrial respiratory chain complex Ⅰ may promote cell migration and invasion by increasing ROS production in colon cancer cells.