Fast exchange of extracellular signals between neurons and astrocytes is crucial for synaptic function. Over the last few decades, different pathways of astroglial release of neuroactive substances have been proposed to modulate neurotransmission. However, their involvement in physiological conditions is highly debated. Connexins, the gap junction forming proteins, are highly expressed in astrocytes and have recently been shown to scale synaptic transmission and plasticity. Interestingly, in addition to gap junction channels, the most abundant connexin (Cx) in astrocytes, Cx43, also forms hemichannels. While such channels are mostly active in pathological conditions, they have recently been shown to regulate cognitive function. However, whether astroglial Cx43 hemichannels are active in resting conditions and regulate basal synaptic transmission is unknown. Here we show that in basal conditions Cx43 forms functional hemichannels in astrocytes from mouse hippocampal slices. We furthermore demonstrate that the activity of astroglial Cx43 hemichannels in resting states regulates basal excitatory synaptic transmission of hippocampal CA1 pyramidal cells through ATP signaling. These data reveal Cx43 hemichannels as a novel astroglial release pathway at play in basal conditions, which tunes the moment-to-moment glutamatergic synaptic transmission.