Connexin 43 (Cx43), a gap junctional protein, regulates osteocyte viability, and modulates mechanical stimulation-induced bone remodeling. However, the underlying mechanisms of its action remain unclear. In the current study, osteocyte-like MLO-Y4 cells were exposed to fluid shear stress (FSS) of 16 (physiological) or 30 (high) dyne/cm(2) for the indicated time points. Cx43 gene (Gja1) was silenced using siRNA or the protein was blocked chemically. The signaling molecules related to osteocyte apoptosis, osteogenesis, or osteoclastogenesis were detected at mRNA or protein levels. The results showed that physiological FSS significantly upregulated Cx43, which further inhibited apoptosis pathways (e.g., caspase-3) and osteoclastogenesis signaling (e.g., RANKL), but activated osteogenesis signaling (Sost/sclerostin). Suppressing Cx43 gene (Gja1) by siRNA or chemically blocking gap junction communication enhanced caspase-3, RANKL, and Sost/sclerostin, which could be restored with physiological FSS over 8 h. In addition, high FSS decreased Cx43 expression and adversely affected signaling molecules compared with physiological FSS. The findings indicate the involvement of Cx43 in mechanotransduction of FSS and in the modulation of mechanical loading-related apoptosis, osteogenesis, and osteoclastogenesis of osteocytes. This may provide a cellular and molecular basis for interpreting the biomechanical mechanism of bone absorption and remodeling.