Recent studies indicate that intestinal cholesterol absorption is a multistep process, which is regulated by multiple genes at the enterocyte level. However, the molecular mechanisms whereby there are gender differences in intestinal cholesterol absorption efficiency and the efficiency of cholesterol absorption increases with age have not yet been fully understood. To explore whether aging increases cholesterol absorption via intestinal sterol transporters, we studied the higher-cholesterol absorbing C57L/J vs. the lower-cholesterol absorbing AKR/J mice at 8 (young adult), 36 (older adult), and 50 (aged) weeks of age. To test the hypothesis that estrogen receptor plays an important regulatory role in cholesterol absorption, we investigated the gonadectomized mice of both genders treated with 17-estradiol-releasing pellets at 0, 3, or 6 µg/day and an antiestrogenic ICI 182,780 at 125 µg/day. We found that hepatic outputs of biliary cholesterol were significantly increased with age and in response to high levels of estrogen. Aging significantly enhances cholesterol absorption by suppressing expression of the jejunal and ileal sterol efflux transporters Abcg5 and Abcg8 and upregulating expression of the putative duodenal and jejunal sterol influx transporter Npc1l1. Estrogen significantly augmented cholesterol absorption mostly due to an upregulated expression of intestinal Npc1l1, Abcg5 and Abcg8 via the intestinal estrogen receptor pathway, which can be fully abolished by the antagonist. We conclude that estrogen receptor activated by estrogen and aging enhance cholesterol absorption by increasing biliary lipid output and mediating intestinal sterol transporters favoring influx of intraluminal cholesterol molecules across the apical membrane of the enterocyte.