Abstract: Objective To investigate the effect of matrine eye drops on the levels of cytokines associated with experimental uveitis in rabbits by regulating toll like receptor 4/nuclear factor-κB(TLR4/NF-κB) pathway. Methods The rabbit model of experimental uveitis was established and randomly divided into model group, tobramycin-dexamethasone eye drops group, low(0.50 g/L), medium(0.75 g/L) and high(1.00 g/L) dose matrine eye drops groups, with 8 rabbits in each group. Another 8 rabbits were selected as control group. After successfully modeling, tobramycin-dexamethasone eye drops group, low, medium and high dose matrine eye drops groups were treated with tobramycin-dexamethasone eye drops and matrine eye drops respectively, rabbits in all groups were given the same dose of saline eye drops for 7 days. Slit lamp was used to observe and evaluate the inflammation of anterior segment of rabbit eyes. Hematoxylin-eosin staining(HE) was used to observe the pathological morphology of the retina of rabbits. Enzyme-linked immunosorbent assay(ELISA) was used the levels of cytokines, tumor necrosis factor-α(TNF-α), interleukin-6(IL-6) and interleukin-1(IL-1) in serum and aqueous humor of rabbits, and Western blot was used to detect the expressions of TLR4/NF-κB pathway proteins TLR4, MyD88 and NF-κB p65 in retina. Results Compared with the control group, the clinical and pathological scores, the levels of IL-6, IL-1 and TNF-α in serum and aqueous humor, and the expressions of TLR4, MyD88 and NF-κB p65 in retina were significantly increased in the model group(all P<0.05). Compared with model group, the clinical and pathological scores, the levels of IL-6, IL-1 and TNF-α in serum and aqueous humor, and the expressions of TLR4, MyD88 and NF-κB p65 in retina tissue in all dose matrine eye drops groups and tobramycin-dexamethasone eye drops group were decreased, and there was a dose-dependent relationship among the dose groups of matrine eye drops(all P<0.05). There was no significant difference between the high dose matrine eye drops groups and tobramycin-dexamethasone eye drops group(P>0.05). Conclusion Matrine eye drops can reduce the levels of cytokines in serum and aqueous humor, alleviate inflammation and improve uveitis symptoms in rabbits, which may be achieved by down-regulating TLR4/NF-κB pathway.