Background: Although the current role of cytokines and neuroinflammation in glaucoma remains obscure, it represents an expanding field in research. The purpose of this study was to analyze cytokines in the aqueous humor (AH) of glaucoma patients and in retinas from an ex vivo glaucoma animal model, to aid in determining the role of neuroinflammation in glaucoma. Methods: AH samples were collected from 20 patients during cataract surgeries (controls: n = 10, age = 70.3 \pm 9.742; glaucoma: n = 10, age: 66.5 \pm 8.073) in Shanghai East Hospital, an affiliate of Tongji University, between September 2018 and March 2019 and analyzed in duplicate by Luminex cytokine polystyrene color bead-based multiplex assay. Retinas from female Sprague-Dawley rats (n = 6) were harvested ex vivo and cultured with or without 60 mmHg of hydrostatic pressure for 24 hours. Retinal ganglion cells (RGCs) were quantified using Brn3a staining. Cytokines in the retina and culture medium were analyzed by rat cytokine array (Abcam). Results: At baseline, patients with primary angle closure glaucoma (PACG) have significantly lower levels of IL-6 and IP-10 and a higher level of PDGF-BB in their AH, compared to the controls. Postoperatively, patients with PACG have significantly higher levels of IL-1ra, IL-13, and MIP-1\alpha and a lower level of IL-6. Elevated hydrostatic pressure led to significant RGC loss in the retina, ex vivo, as well as the upregulation of ciliary neurotrophic factor (CNTF), IL-6, IL-10, IL-4, and TIMP-1 alongside the downregulation of PDGF-AA, MMP-8, TNF-\alpha, and IFN-\gamma. Furthermore, eight cytokines were detected as being downregulated in the culture medium, including PDGF-AA, MMP-8, and IL-4. Conclusions: Proinflammatory cytokines showed changes in both AH and ex vivo. Further studies are needed on the role of these cytokines and their corresponding signaling pathways in both neurodegeneration and glaucoma.