Guinea pigs were fed a hypercholesterolemic diet (0.25 g/100 g cholesterol) and randomly allocated either to a Control group (n = 9) or to a Lutein (0.1 g/100 g) group (n = 10) for 12 weeks to evaluate oxidative stress and inflammation in both liver and eyes. Malondialdehyde (MDA) concentrations and inflammatory cytokines were measured as well as hepatic nuclear factor-kappaB (NF-κB) binding. Lutein concentrations were greater in eyes ( P < 0.01) and liver ( P < 0.001) in the Lutein group. All guinea pigs had high concentrations of hepatic cholesterol as well as high plasma ALT and AST levels indicative of liver injury. However, the Lutein group had 43% lower hepatic free cholesterol than the Controls ( P < 0.05). Hepatic MDA and MDA in the eye were lower in the Lutein compared to the Control group ( P < 0.05). Hepatic tumor necrosis factor-α was 32% lower in the Lutein group ( P < 0.05). Lastly, the Lutein group presented lower NF-κB DNA binding activity than the Control group ( P < 0.001). These results suggest that in the presence of high cholesterol, lutein exerts both antioxidant and anti-inflammatory effects, which can be explained by attenuated NF-κB DNA binding activity. Furthermore, results also suggest that lutein accumulates in the eyes of guinea pigs to protect against oxidative stress.