Thrombin is a multifunctional protease that can activate hemostasis and coagulation through the cleavage of fibrinogen to form fibrin clots. Thrombin also plays a crucial role in migration and metastasis of human cancer cells. However, the effect of thrombin on migration activity in human chondrosarcoma cells is mostly unknown. Here, we found that thrombin increased the migration and expression of matrix metalloproteinase (MMP)-2 and MMP-13 in human chondrosarcoma cells (JJ012 and SW1353 cells). By using pharmacological inhibitors or activators or genetic inhibition by the protease-activated receptor (PAR), we found that the PAR1 and PAR4 receptor but not PAR3 receptor are involved in thrombin-mediated cell migration and MMPs expression. Thrombin-mediated migration and MMPs up-regulation was attenuated by phospholipase C (PLC), protein kinase C, and c-Src inhibitor. Activations of PLCbeta, PKCalpha, c-Src, and NF-kappaB pathways after thrombin treatment was demonstrated, and thrombin-induced MMPs expression and migration activity was inhibited by the specific inhibitors and mutants of PLC, PKC, c-Src, and NF-kappaB cascades. Taken together, our results indicated that thrombin enhances the migration of chondrosarcoma cells by increasing MMP-2 and MMP-13 expression through the PAR/PLC/PKCalpha/c-Src/NF-kappaB signal transduction pathway.