Guanylyl cyclase-B (GC-B) is a receptor for C-type natriuretic peptide, which regulates proliferation and migration in fibroblasts in various cells. Here we report that GC-B activity is suppressed by sphingosine-1-phosphate (S1P) specifically through the type 2 S1P receptor (S1P₂) in Swiss 3T3 fibroblasts and the type 2 and 3 (S1P₃) receptors in HeLa cells. In Swiss 3T3 fibroblasts, S1P-dependent suppression of GC-B activity was not mimicked by an S1P₁-specific agonist, and was abrogated by an S1P₂-specific, but not an S1P₃-specific, antagonist. In HeLa cells, siRNA-mediated depletion of either S1P₂ or S1P₃ reduced S1P-dependent suppression of GC-B activity, whereas depletion of S1P₁ had no effect. S1P-mediated suppression of GC-B activity was blocked by the Rho inhibitor, Clostridium difficile toxin B, and by expression of a dominant-negative RhoA mutant, but not by Rho kinase (ROCK) inhibitors. Toxin B treatment also reduced S1P-induced GC-B dephosphorylation, an established regulatory mechanism of GC-B activity. Furthermore, S1P-dependent suppression of GC-B activity was partially blocked by protein phosphatase 1 and 2A inhibitors. These results are consistent with a mechanism whereby S1P, acting through S1P₂ and/or S1P₃, inhibits GC-B activity via a Rho-dependent but ROCK-independent pathway that involves activation of a subset of protein phosphatases.