Astrocytes interact dynamically with neurons by modifying synaptic activity and plasticity. This interplay occurs through a process named gliotransmission, meaning that neuroactive molecules are released by astrocytes. Acting as a gliotransmitter, D-serine, a co-agonist of the NMDA receptor at the glycine-binding site, can be released by astrocytes in a calcium [Ca]-dependent manner. A typical feature of astrocytes is their high expression level of connexin43 (Cx43), a protein forming gap junction channels and hemichannels associated with dynamic neuroglial interactions. Pharmacological and genetic inhibition of Cx43 hemichannel activity reduced the amplitude of NMDA EPSCs in mouse layer 5 prefrontal cortex pyramidal neurons without affecting AMPA EPSC currents. This reduction of NMDA EPSCs was rescued by addition of D-serine in the extracellular medium. LTP of NMDA and AMPA EPSCs after high-frequency stimulation was reduced by prior inhibition of Cx43 hemichannel activity. Inactivation of D-serine synthesis within the astroglial network resulted in the reduction of NMDA EPSCs, which was rescued by adding extracellular D-serine. We showed that the activity of Cx43 hemichannels recorded in cultured astrocytes was [Ca] dependent. Accordingly, in acute cortical slices, clamping [Ca] at a low level in astroglial network resulted in an inhibition of NMDA EPSC potentiation that was rescued by adding extracellular D-serine. This work demonstrates that astroglial Cx43 hemichannel activity is associated with D-serine release. This process, occurring by direct permeation of D-serine through hemichannels or indirectly by Ca entry and activation of other [Ca]-dependent mechanisms results in the modulation of synaptic activity and plasticity. We recorded neuronal glutamatergic (NMDA and AMPA) responses in prefrontal cortex (PFC) neurons and used pharmacological and genetic interventions to block connexin-mediated hemichannel activity specifically in a glial cell population. For the first time in astrocytes, we demonstrated that hemichannel activity depends on the intracellular calcium concentration and is associated with D-serine release. Blocking hemichannel activity reduced the LTP of these excitatory synaptic currents triggered by high-frequency stimulation. These observations may be particularly relevant in the PFC, where D-serine and its converting enzyme are highly expressed.