The brain is the most cholesterol-rich organ in the body and consists of 25% of total cholesterol. ATP-binding cassette (ABC) transporters play essential roles in cellular cholesterol efflux and homeostasis in the brain. Recently, ABCG1, ABCG4 and ABCA1 expression in the adult brain has been described. The absence of one or more of these transporters has been implicated in the development of neurodegenerative diseases. In this study, we characterized the mRNA and protein expression levels of ABCG1, ABCG4 and ABCA1 in the developing postnatal brain of normal C57BL/6J mice fed a chow diet. We studied the correlation between ABC transporters expression and cholesterol levels (free cholesterol, esterified cholesterol) in the brain and serum, to elucidate a potential role of these transporters in cholesterol metabolism in the brain and body during postnatal stages. We further investigated the changes of expression levels of ABCG1, ABCG4, ABCA1, cholesterol related genes and brain cholesterol levels in ABCG1-/-, ABCG4-/- and ABCG1-/- ABCG4-/- double knockout mice. ABCA1 mRNA expression were detectable in multiple tissues, and ABCG1, ABCG4 were highly expressed in adult brain. ABCG1 and ABCG4 mRNA levels peaked at 42 days of age, while ABCA1 mRNA levels were near baseline. ABCG1 protein levels peaked at day 28 then decreased, while ABCG4 levels peaked at day 42. ABCA1 levels remained near baseline. Interestingly, circulating plasma and brain esterified cholesterol levels exhibited a biphasic distribution, which peaked at day 42. Loss of ABCG1 is compensated by increased ABCG4 and vice versa. Loss of both ABCG1 and ABCG4 results in altered expression of cholesterol synthesis related genes and cholesterol accumulation in the brain. The data suggest that ABCG1 and ABCG4, but not ABCA1 are important for transporter function in the developing brain. ABCG1 and ABCG4 play complementary roles in maintaining brain cholesterol homeostasis.