Our aim was to study the effect of 9-cis β-carotene-rich powder of the alga Dunaliella bardawil on lipid profile, atherogenesis, and liver steatosis in high-fat diet-fed LDL receptor knockout mice. In 4 sets of experiments, mice were distributed into the following groups: control, fed an unfortified diet; Dunaliella 50, fed a diet composed of 50% 9-cis and 50% all-trans β-carotene; Dunaliella 25, fed a diet containing 25% 9-cis and 75% all-trans β-carotene; β-carotene-deficient Dunaliella, fed β-carotene-deficient Dunaliella powder; and all-trans β-carotene, fed a synthetic all-trans β-carotene. All fortified diets contained 0.6% total β-carotene. Algal 9-cis β-carotene was absorbed by the mice and accumulated in the liver. Synthetic all-trans β-carotene was not converted to 9-cis β-carotene. Dunaliella 50 inhibited high-fat diet-induced plasma cholesterol elevation by 40-63% and reduced cholesterol concentrations in the atherogenic VLDL and LDL. Atherosclerotic lesion area in mice treated with Dunaliella 50 was 60-83% lower compared with mice fed the high-fat diet alone. β-Carotene-deficient Dunaliella did not influence plasma cholesterol and atherogenesis, suggesting that β-carotene is essential for a Dunaliella protective effect. Moreover, by administrating Dunaliella powder containing different levels of 9-cis and all-trans β-carotene isomers, we found that the effect on plasma cholesterol concentration and atherogenesis is 9-cis-dependent. Dunaliella 50 also inhibited fat accumulation and inflammation in the livers of mice fed a high-fat diet, which was accompanied by reduced mRNA levels of inflammatory genes. These results in mice suggest that 9-cis β-carotene may have the potential to inhibit atherogenesis in humans.