Post-traumatic stress disorder (PTSD) is a psychiatric illness that develops after a person is exposed to an extremely stressful event. It can be understood as maladaptive responses due to deregulation of the fear and anxiety brain circuits, in which the amygdala and hippocampus play a fundamental role. Astrocytes are known to have an active role in synaptic function, with the release of gliotransmitters being a critical aspect of the functioning of tripartite synapses. Recent experimental studies demonstrated the role played by astrocytic Cx43 hemichannels in the release of gliotransmitters and their participation in emotional processing. The aim of this research was to evaluate the functionality of astrocytic Cx43 hemichannels in a model of post-traumatic stress, known as single prolonged stress (SPS). In this work, we applied SPS to adult mice and evaluated their anxious behavior by performing behavioral tests (elevated plus maze and open field). Then, we measured hemichannel activity through an ethidium bromide uptake assay in ex vivo tissue. For staining validation, we assessed bromide uptake in the presence of a synthetic peptide, which selectively blocks Cx43 hemichannels. GFAP immunohistochemistry was used to identify astroglial cells. Changes in Cx43 hemichannel activity in astrocytes might represent a novel mechanism in PTSD and a new target for pharmacotherapy.