Vitamin A deficiency (VAD) is a major health problem, especially in developing countries. In this study, we investigated the effect of VAD from weaning to adulthood in apoE^−/− mice. Three-week-old male mice were allocated into four diet groups: I. VAD II. VAD+vitamin A (VA), 1500 IU retinyl-palmitate; III. VAD+β-carotene (BC), 6 g/kg feed, containing 50% all-trans and 50% 9-cis BC. IV. VAD with BC and VA (BC+VA). After 13 weeks, we assessed the size of atherosclerotic plaques and measured VA in tissues and BC in plasma and tissues. VAD resulted in diminished hepatic VA levels and undetectable brain VA levels compared to the other groups. BC completely replenished VA levels in the liver, and BC+VA led to a two-fold elevation of hepatic VA accumulation. In adipose tissue, mice fed BC+VA accumulated only 13% BC compared to mice fed BC alone. Atherosclerotic lesion area of BC group was 73% lower compared to VAD group (p < 0.05). These results suggest that BC can be a sole source for VA and inhibits atherogenesis.