Connexin and pannexin membrane channel proteins for gap junctions which are conduits that allow neuronal, glial, and vascular tissues interactions. In the brain, this interaction is highly critical for homeostasis and brain repair after injury. The main gap junction protein in the brain, Connexin43 (Cx43), is mainly present in astrocytes; its function is influenced by kinases that phosphorylate specific serine sites located near its C-terminus. Stroke is a powerful inducer of kinase activity, but its effect on Cx43 is unknown. We investigated the impact of a permanent middle cerebral artery occlusion (MCAO) stroke model in mice that were wild-type (WT) or knock-in of Cx43 with serine to alanine mutations at the protein kinase-C site Cx43S368A (PKC), the casein kinase-1 sites Cx43S325A/328Y/330A (CK1) and the mitogen-activated protein kinase sites Cx43S255/262/279/282A (MK4). We demonstrate that MK4 transgenic animals exhibit a significant decrease in infarct volume which was associated with significant behavioral improvement. An increase in astrocyte reactivity with a concomitant decrease in microglial reactivity was observed in MK4 mice. In contrast to WT, MK4 astrocytes displayed reduced Cx43 hemichannel activity. To further validate the potential of targeting Cx43 hemichannels in stroke, pharmacological blockade of Cx43 hemichannels with TAT-Gap19 significantly decreased infarct volume in WT animals. This study provides novel molecular insights and charts new avenues for therapeutic intervention associated with Cx43 function. Understanding the molecular mechanisms by which these membrane channels function, in health and disease, might be particularly influential in establishing conceptual frameworks to develop new therapeutics against connexin and pannexin channels.