Hypothalamic dysfunction is a key pathological factor in inflammation-associated depression. In the present study, isobaric tags for relative-absolute quantitation (iTRAQ) combined with mass spectrometry were employed to detect the proteomes in the hypothalamus of the lipopolysaccharide (LPS)-induced depression mouse. A total of 187 proteins were differentially expressed compared with the control group. The results indicated altered molecules were clustered into Ephrin receptor signalling; glutamatergic transmission, and inflammation-related signalling. Ephrin type-B receptor 2 (EPHB2), a transmembrane receptor protein in Ephrin receptor signalling, was significantly elevated and interacted with the accumulated NMDAR subunit GluN2A in the hypothalamus. Additionally, molecules involved in synaptic plasticity regulation, such as hypothalamic postsynaptic density protein-95 (PSD-95), p-AKT and brain-derived neurotrophic factor (BDNF), were significantly altered in the LPS-induced depressed group. It might be an underlying pathogenesis that the EPHB2-GluN2A-AKT cascade regulates synaptic plasticity in depression. EPHB2 can be a potential therapeutic target in the correction of glutamatergic transmission dysfunction. In summary, our findings point to the previously undiscovered molecular underpinnings of the pathophysiology in the hypothalamus of inflammation-associated depression and offer potential targets to develop antidepressants.