Our previous studies have shown that the early components of the classical complement pathway protect low-density lipoprotein receptor deficient mice (Ldlr−/−) from atherogenesis when fed a low-fat diet. However, the role of the alternative pathway remains unknown. To investigate this, we crossed mice lacking the alternative pathway activator Factor B (Bf−/−) with Ldlr−/− mice. Lipid profiles after 12 weeks on a high-fat diet showed significantly reduced levels of total cholesterol (25.2%±0.9 mmol/l, n=14, vs 40.5%±1.8 mmol/l, n=17, p<0.0001) and lipoproteins (∼1.5-fold lower VLDL and LDL) in the Bf−/−.Ldlr−/− mice compared with the Ldlr−/− animals. Consistent with this, high-fat fed Bf−/−.Ldlr−/− mice had decreased cross-sectional aortic root lesion fraction area (median 12.2%, IQR 8.92% to 15.82% vs 16.3%, 14.76% to 19.74%, p=0.0016) and reduced lesion complexity. These changes were associated with reduced complement activation in the circulation and in atherosclerotic plaques. There was no difference in lipid profiles between Bf−/−.Ldlr−/− and Ldlr−/− mice fed a low-fat diet, but in these groups administration of lipopolysaccharide (LPS) led to significant increase in atherosclerosis only in Ldlr−/− and not in Bf−/−.Ldlr−/− (aortic root lesion fraction: Bf−/−.Ldlr−/−: 7.85%, range 4.93 to 15.24%, n=9; Ldlr−/−: 22.53%, range 17.36 to 25.25%, n=10, p=0.0009), indicating that the alternative pathway also has a key role in endotoxin-mediated exacerbation of disease. These data indicate that amplification of complement activation by the alternative pathway in response to diet or infection may convert the complement system from a protective to a more atherogenic role.