Astrocytes are known to regulate and support neuronal and synaptic functions. Changes in their size and morphology in mouse models result in mental retardation. However, the mechanism underlying these morphological changes remains unclear. In the present study, abnormal astrocyte morphology was found in the mouse brain following knockout of dendritic cell factor 1 (Dcf1). Immunoprecipitation-mass spectrometry (IP-Mass) identified that ATP1B1 is bound to DCF1, and co-immunoprecipitation and cell fluorescence further confirmed an interaction between these two proteins, with asparagine residue 266 of ATP1B1 being required for the interaction with DCF1. Moreover, Dcf1 knockout in mice resulted in upregulation of ATP1B1 expression in the hippocampus. Furthermore, DCF1 interaction with ATP1B1 in astrocytes impaired their structural plasticity. Ultimately, Dcf1 knockout increased glutamate release. Mechanism exploration proposed that Dcf1 knockout led to significantly perturbed expression of AMPA receptors (AMPARs) and induced morphological changes in astrocytes through the P38 signaling pathway. Our data shed light on the possible mechanisms underlying changes in astrocyte morphology and provide new avenues for the exploration of proteins involved in glutamate release.