Purpose Plasmalogens are glycerophospholipids containing a vinyl‐ether bond at sn‐1 position of their glycerol backbone and polyunsaturated fatty acids (PUFAs) at sn‐2. We have previously shown that plasmalogens are involved in the regulation of perinatal retinal vascular development and particularly in astrocyte template formation (Saab et al, PLoSONE 2012 9(6):e101076). Since retinal Müller cells and astrocytes can communicate through calcium waves and connexin 43‐rich gap junctions, the aim of our study was to determine whether a reduction of plasmalogen levels affects communication between retinal glial cells. Methods Primary Müller cells and astrocyte were isolated from retinas of 9‐days old and cerebral cortex from 21‐days old Wistar rats, respectively. SiRNA against DHAPA‐AT ‐the key enzyme of plasmalogen biosynthesis‐ were used to decrease plasmalogen content of Müller cells before they were co‐cultured with naïve astrocytes. Calcium waves initiated in Müller cells by topical application of ATP were monitored by Fura‐2 calcium imaging. Expression levels of cellular proteins involved in calcium metabolism, including connexin 43, were evaluated by proteomics and Western‐blotting in reponse to Si RNA treatment. Results Treating Müller cells with SiRNA against DHAP‐AT led to a reduction of plasmalogen content of about 60% and to a remodelling of PUFA distribution in cell membranes. The expression of connexion 43 was reduced by more than 50% in Müller cells treated with SiRNA. This was associated with an alteration of calcium waves originating from Müller cells as well as a slowed propagation to neighbouring astrocytes. Conclusions These data suggest that membrane plasmalogens content influences cell‐to‐cell communication between retinal Müller cells and astrocytes. Altered coupling between these cells might be one of the factors influencing perinatal vascular development.