Parathyroid hormone (PTH) binding to its receptor activates protein kinase A (PKA), protein kinase C (PKC) and calcium signaling to induce transcription of primary response genes (PRGs) in osteoblasts. Adenovirus E4 promoter-binding protein/nuclear factor regulated by IL-3 (E4BP4/NFIL3), a transcriptional repressor, is a PTH-induced PRG in primary mouse osteoblasts (MOBs). Here we investigate the signaling pathway(s) that lead to PTH induction of E4bp4 mRNA expression. Ten and 100 nM PTH induced maximum E4bp4 expression in MOBs. Forskolin (FSK), an adenylate cyclase inducer, 8-bromo-cAMP, a cAMP analog, and phorbol myristate acetate (PMA), a PKC activator, increased E4bp4 mRNA levels, whereas ionomycin, a calcium ionophore, had no effect. Pretreatment of cells with 30 µM H89, a PKA inhibitor, strongly inhibited PTH and FSK induced E4bp4 expression. In contrast, overnight pretreatment with 1 µM PMA to down-regulate PKC signaling did not alter PTH and FSK effects. Moreover, PTH (3-34) that does not activate cAMP signaling did not increase E4bp4 expression. PGE_2, which signals through cAMP, increased E4bp4 mRNA at all doses, while PGF_2a that primarily activates PKC and calcium signaling, induced E4bp4 only at high doses and fluprostenol that only activates PKC and calcium signaling, had no effect. Finally, 80 µg/kg PTH (1-34) ip injection induced E4bp4 mRNA expression at 1 h in mice. In contrast, 80 µg/kg PTH (3-34) had no affect. Our data suggest that PTH-induced E4bp4 mRNA expression is mediated primarily through cAMP-PKA signaling in vitro and in vivo. In conjunction with our previous report, we hypothesize that E4bp4 attenuates transcription of osteoblastic genes possessing E4bp4 promoter binding sites.