Connective tissue growth factor (CTGF), a member of the Cyr 61, CTGF, Nov (CCN) family of proteins, regulates multiple cellular functions. Overexpression of CTGF in vivo causes osteopenia, but in vitro CTGF can induce osteoblastogenesis. To investigate mechanisms involved in the effects of CTGF on osteoblastic cell differentiation, we examined whether CTGF modifies the activity of nuclear factor of activated T-cells (NFATc) 1, a transcription factor that cooperates with osterix in the formation of new bone. CTGF increased the transactivation of a transiently transfected reporter construct, where 9 NFAT binding sites direct the expression of luciferase (9xNFAT-Luc) and the activity of the Regulators of calcineurin 1 exon 4 (Rcan1.4) promoter, an NFAT target gene. We postulated that CTGF could modify the phosphorylation of NFAT by regulating glycogen synthase kinase 3β (GSK3β). CTGF increased the mRNA levels of Protein kinase cyclic guanosine monophosphate (cGMP) dependent type II (Prkg2), the gene encoding for cGMP dependent protein kinase II (cGKII) which phosphorylates GSK3β. Accordingly, CTGF induced GSK3β phosphorylation and decreased the active pool of GSK3β, a kinase that phosphorylates NFAT and leads to its nuclear export. As a consequence, CTGF favored the nuclear localization of NFATc1. Downregulation of PRKG2 by RNA interference reversed the effect of CTGF on the transactivation of the 9xNFAT reporter construct and the Rcan 1.4 promoter, confirming the role of cGKII in the activation of NFAT by CTGF. In conclusion, CTGF enhances NFAT signaling through the induction of cGKII and the phosphorylation of GSK3β. J. Cell. Biochem. 110: 477–483, 2010. © 2010 Wiley-Liss, Inc.