Gap junction intercellular communication in osteocytes plays an important role in bone remodeling in response to mechanical loading;​ however, the responsible molecular mechanisms remain largely unknown. Here, we show that phosphoinositide-3 kinase (PI3K)/Akt signaling activated by fluid flow shear stress and prostaglandin E₂ (PGE₂) had a stimulatory effect on both connexin 43 (Cx43) mRNA and protein expression. PGE₂ inactivated glycogen synthase kinase 3 (GSK-3) and promoted nuclear localization and accumulation of β-catenin. Knockdown of β-catenin expression resulted in a reduction in Cx43 protein. Furthermore, the chromatin immunoprecipitation (ChIP) assay demonstrated an association of β-catenin with the Cx43 promoter, suggesting that β-catenin could regulate Cx43 expression at the level of gene transcription. We have previously reported that PGE₂ activates cyclic AMP (cAMP)-protein kinase A (PKA) signaling and increases Cx43 and gap junctions. Interestingly, the activation of PI3K/Akt appeared to be independent of the activation of PKA, whereas both PI3K/Akt and PKA signaling inactivated GSK-3 and increased β-catenin translocation. Together, these results suggest that shear stress, through PGE₂ release, activates both PI3K/Akt and cAMP-PKA signaling, which converge through the inactivation of GSK-3, leading to the increase in nuclear accumulation of β-catenin. β-Catenin binds to the Cx43 promoter, stimulating Cx43 expression and functional gap junctions between osteocytes.