Matrix metalloproteinase-9 (MMP-9) plays a crucial role in cell invasion and cancer metastasis. In this study, we showed that cholic acid (CA), a major primary bile acid, can induce MMP-9 expression in colon cancer HT29 and SW620 cells. CA increased reactive oxygen species (ROS) production and also activated phosphorylation of ERK1/2, JNK, and p38 MAPK. Specific inhibitors and mutagenesis studies showed that ERK1/2 and JNK functioned as upstream signals in the activation of AP-1, and p38 MAPK functioned as an upstream signal in the activation of NF-κB. N-acetyl-L-cysteine (NAC, an ROS scavenger) and diphenyleneiodonium chloride (DPI, an NADPH oxidase inhibitor) inhibited CA-induced activation of ERK1/2, JNK, and p38 MAPK, indicating that ROS production by NADPH oxidase could be the furthest upstream signal in MMP-9 expression. Colon cancer cells pretreated with CA showed remarkably enhanced invasiveness. Such enhancement was partially abrogated by MMP-9-neutralizing antibodies. These results demonstrate that CA could induce MMP-9 expression via ROS-dependent ERK1/2, JNK-activated AP-1, and p38-MAPK-activated NF-κB signaling pathways, which in turn stimulate cell invasion in human colon cancer cells.