Metastasis is the primary cause of cancer-associated mortality. The ternary IPP complex of integrin-linked kinase, PINCH and parvin functions as a signaling platform for integrins, which modulate numerous cellular processes including cell migration and invasion. Chelidonine, isolated from Chelidonium majus, is a benzophenanthridine alkaloid that exhibits anticancer properties; however, the anti-migratory and anti-invasive effects of chelidonine remain unknown. The aim of the present study was to investigate the inhibitory effects of chelidonine on migration and invasion of MDA-MB-231 human breast cancer cells, and to determine the underlying mechanisms. Chelidonine was shown to inhibit the migration and invasion of MDA-MB-231 cells in a concentration-dependent manner, without affecting the cell viability. Chelidonine did not significantly inhibit the adhesion of the cells to type 1 collagen (COL-I), however it did affect cell spreading and reorganization of the actin cytoskeleton. Chelidonine also inhibited COL-I-induced protein kinase B (Akt) activation and translocation to the plasma membrane, however, it did not significantly inhibit the activation of focal adhesion kinase. Notably, chelidonine treatment significantly inhibited COL-I-induced formation of the IPP complex and activation of IPP downstream signaling molecules, such as extracellular signal-regulated kinase (ERK)1/2. These results suggest that chelidonine exhibits anti-migratory and anti-invasive effects in MDA-MB-231 cells, by suppressing COL-I-induced integrin signaling, through inhibiting the formation of the IPP complex and subsequent down-regulation of IPP downstream signaling molecules, such as Akt and ERK1/2. These results suggest that chelidonine may be a potential therapeutic agent against metastasis of invasive human cancer cells.