In anaplastic thyroid cancer C643 cells, sphingosine 1-phosphate (S1P) attenuates migration by activating the S1P2 receptor and the Rho-ROCK pathway. In the present study, we show that stimulating C643 cells with S1P decreases the expression, secretion and activity of matrix metalloproteinase-2 (MMP2), and to a lesser extent MMP9. Using receptor-specific antagonists, and S1P2 siRNA, we showed that the inhibition of expression of MMP2 is mediated through S1P2. Furthermore, S1P inhibited calpain activity, and inhibiting calpain pharmacologically, inhibited the effect of S1P on MMP2 expression and activity, and on MMP9 activity. S1P treatment increased Rho activity, and by incubating cells with the Rho inhibitor C3 transferase or the ROCK inhibitor Y27632, the S1P-induced inhibition of invasion and MMP2 expression and activity was abolished. We conclude that S1P attenuates the invasion of C643 cells by activating S1P2 and the Rho-ROCK pathway, by decreasing calpain activity, and by decreasing the expression, secretion and activity of MMP2 and, to a lesser extent, MMP9. Our results thus unveil a novel function for the S1P2 receptor in attenuating thyroid cancer cell invasion.