Connective tissue growth factor (CTGF/CCN2) is an immediate early gene-encoded polypeptide modulating cell growth and collagen synthesis. The importance of CTGF/CCN2 function is highlighted by its disregulation in fibrotic disorders. In this study, we investigated the regulation and signaling pathways that are required for various stimuli of intracellular signaling events to induce the expression of the endogenous CTGF/CCN2 gene in smooth muscle cells. Incubation with the bioactive lysolipid sphingosine 1-phosphate (S1P) produced a threefold increase, whereas stimulation with either fetal bovine serum or anisomycin induced an even stronger activation (eightfold) of CTGF/CCN2 expression. Using a combination of pathway-specific inhibitors and mutant forms of signaling molecules, we found that S1P- and fetal bovine serum-induced CTGF/CCN2 expression were dependent on both RhoA GTPase and p38 mitogen-activated protein kinase transduction pathways, whereas the effects of anisomycin largely involved p38 and phosphatidyl inositol 3-kinase signaling mechanisms. However, activation via these signaling events was absolutely dependent on actin cytoskeleton integrity. In particular, RhoA-dependent regulation of the CTGF/CCN2 gene was concomitant to increased polymerization of actin microfilaments resulting in decreased G- to F-actin ratio and appeared to be achieved at the transcriptional level. The p38 signaling pathway was RhoA-independent and led to CTGF/CCN2 mRNA stabilization. Use of actin-binding drugs showed that the actual physical state of monomeric G-actin is a critical determinant for CTGF/CCN2 gene induction. These data indicate that distinct cytoskeletally based signaling events within the intracellular signaling machinery affect either transcriptionally or post-transcriptionally the expression of the CTGF/CCN2 gene in smooth muscle cells.