This study sought to explore the role of matrine in the metastasis of cancer cells and to gain insight into the possible mechanism of matrine's ability to inhibit cancer metastasis. Accordingly, changes in vasodilator-stimulated phosphoprotein (VASP) phosphorylation and in cAMP-dependent protein kinase (PKA) activity during cell detachment and reattachment were first investigated. After administration of matrine (50 μg/ml), the decrease in VASP phosphorylation paralleled the decrease in PKA activity. Matrine was found to significantly inhibit HeLa cell adhesion to collagen I. To determine the effect of matrine on the migration of HeLa cells, we analysed the migratory behaviour of HeLa cells in a two and three-dimensional cell migration assay. In a two-dimensional cell migration assay, the average cell migration velocity was significantly reduced by matrine compared with the control. Moreover, in a three-dimensional cell migration assay performed with the Transwell system, HeLa cells treated with matrine (50 μg/ml) were found to migrate less than the control cells. These data suggest that the inhibitory effect of matrine may be produced by decreased phosphorylation of VASP due to inhibition of the activity of PKA during HeLa cell adhesion and migration.