There is evidence that astroglial connexin43 (Cx43) in the supraoptic nucleus (SON) is required for the hyperosmolarity‑induced increase in Fos protein expression in magnocellular neurosecretory cells (MNCs). In the present study, the role of astroglial Cx43 in the synthesis and release of vasopressin (VP) by MNCs in the SON subjected to hyperosmotic stimulus was examined. The results revealed that the VP levels in the SON and plasma were increased following acute hyperosmotic stimulus. Treatment of MNCs with Cx43‑specific antisense oligodeoxynucleotides (ASODN), which temporarily reduced Cx43 protein production, limited the VP synthesis and release induced by a hyperosmotic stimulus. Similarly, the addition of gap junction and Cx43 hemichannel blockers also attenuated the VP synthesis and release induced by an acute hyperosmotic stimulus. A high extracellular [Ca2+]([Ca2+]o) has been demonstrated to reduce the gap junction activity or opening probability of Cx54 hemichannels. Notably, it was identified that high [Ca2+]o attenuated the VP synthesis and release induced by acute hyperosmotic stimulus, while low [Ca2+]o had a weak or no effect. These results suggested that Cx43 participates in the VP synthesis and release induced by hyperosmotic stimulation in the SON.