This study investigated the anti-inflammatory effects of water-dispersible hesperetin (WD-Hpt) in an endotoxin-induced uveitis (EIU) rat model. The rats were orally administered 10, 25, or 50 mg/kg WD-Hpt immediately after lipopolysaccharide (LPS) injection at the concentration of 200 μg. Clinical scores, cellular inflammation, the aqueous humor (ApH) protein concentration, as well as the levels of tumor necrosis factor (TNF)-α, cyclooxygenase (COX)-2 and inducible NO synthase (iNOS) in AqH, and histopathological grades were assessed. Immunohistostaining and mRNA analyses measured expressions of TNF-α, COX-2, iNOS, activated nuclear factor (NF)-κB p65, I kappa B (IκB)-α degradation, phosphorylated (p)-IκB kinase (IKK) α/β, β-catenin, and glycogen synthase kinase (GSK)-3β. Compared to LPS treated group (LPS txg), WD-Hpt treatment groups (WD-Hpt txg) resulted in the following results: 1) clinical scores improved [LPS txg
3.90 ± 0.20, WD-Hpt txg
2.40 ± 0.37 (P<
0.05)], 2) the number of inflammatory cells in AqH decreased [LPS txg
8.65 ± 1.41 × 105 cells/mL, WD-Hpt txg
3.83 ± 1.20 × 105 cells/mL (P<
0.05)], 3) AqH protein concentration reduced [LPS txg
36.65 ± 2.71 mg/mL, WD-Hpt txg
28.73 ± 2.36 mg/mL (P<
0.05)], and 4) decreased levels of TNF-α [LPS txg
69.55 ± 7.38 pg/mL, WD-Hpt txg
35.18 ± 9.22 pg/mL (P<
0.001)], iNOS [LPS txg
153.37 ± 12.72 μM, WD-Hpt txg
110.79 ± 13.27 μM (P<
0.05)], and COX-2 [LPS txg
1,080.56 ± 196.06 pg/mL, WD-Hpt txg
477.80 ± 66.61 pg/mL (P<
0.01)] in AqH were observed, and histopathological grades improved [LPS txg
2.80 ± 0.40, WD-Hpt txg
1.50 ± 0.50 (P<
0.05)]. Immunostaining and mRNA analysis revealed that 50 mg/kg WD-Hpt effectively suppressed iNOS, COX-2, NF-κB p65, IκB-α degradation, p-IKKα/β, β-catenin, and GSK-3β expression. These findings suggested that WD-Hpt exerts anti-inflammatory effects by targeting the NF-κB and Wnt/β-catenin pathways.