Connective Tissue Growth Factor (CCN2/CTGF) mediates TGFβ induced fibrosis. Drug-induced gingival overgrowth is tissue specific. Here the role of the phosphoinositol 3 kinase (PI3K) pathway in mediating TGFβ1 stimulated CCN2/CTGF expression in primary human adult gingival fibroblasts and human adult lung fibroblasts was compared. Data indicate that PI3K inhibitors attenuate up-regulation of TGFβ1-induced CCN2/CTGF expression in human gingival fibroblasts independent of reducing JNK MAP kinase activation. Pharmacologic inhibitors and siRNA mediated knockdown studies indicate that calcium-dependent isoforms and an atypical isoform of protein kinase C (PKC) do not mediate TGFβ1 stimulated CCN2/CTGF expression in gingival fibroblasts. As glycogen synthase kinase-3-beta (GSK3β) can undergo phosphorylation by the PI3K/pathway, effects of GSK3β inhibitor kenpaullone and siRNA knockdown were investigated. Data in gingival fibroblasts indicate that kenpaullone attenuates TGFβ1 mediated CCN2/CTGF expression. Activation of the Wnt canonical pathways with Wnt3a, which inhibits GSK3β, similarly inhibits TGFβ1 stimulated CCN2/CTGF expression. In contrast, inhibition of GSK3β by Wnt3a does not inhibit, but modestly stimulates CCN2/CTGF levels in primary human adult lung fibroblasts and is β-catenin-dependent, consistent with previous studies performed in other cell models. These data identify a novel pathway in gingival fibroblasts in which inhibition of GSK3β attenuates CCN2/CTGF expression. In adult lung fibroblasts inhibition of GSK3β modestly stimulates TGFβ1 regulated CCN2/CTGF expression. These studies have potential clinical relevance to the tissue-specificity of drug-induced gingival overgrowth.