The process of cholesterol absorption has yet to be completely defined at the molecular level. Because of its ability to esterify cholesterol for packaging into nascent chylomicrons, ACAT2 plays an important role in cholesterol absorption. However, it has been found that cholesterol absorption is not completely inhibited in ACAT2-deficient (ACAT2 KO) mice. Because ABCA1 mRNA expression was increased 3-fold in the small intestine of ACAT2 KO mice, we hypothesized that ABCA1-dependent cholesterol efflux sustains cholesterol absorption in the absence of ACAT2. To test this hypothesis, cholesterol absorption was measured in mice deficient in both ABCA1 and ACAT2 (DKO). Compared with wild-type, ABCA1 KO, or ACAT2 KO mice, DKO mice displayed the lowest level of cholesterol absorption. The concentrations of hepatic free and esterified cholesterol and gallbladder bile cholesterol were significantly reduced in DKO compared with wild-type and ABCA1 KO mice, although these measures of hepatic cholesterol metabolism were very similar in DKO and ACAT2 KO mice. We conclude that ABCA1, especially in the absence of ACAT2, can have a significant effect on cholesterol absorption, although ACAT2 has a more substantial role in this process than ABCA1.