Estrogen is considered to be involved in duodenal function; however, the details of its receptor expression are largely unknown. The purpose of this study was to determine the expression and localization of estrogen receptors (ERs) in mouse duodenum. Male and female C57BL/6J mouse intestinal tissues were used to investigate the expression of ERα and ERβ by RT-PCR, western blotting, immunohistochemistry, and Southwestern histochemistry. ERβ, but not ERα, was expressed in proximal duodenal epithelium, but not in jejunum and ileum. The expression of ERβ mRNA and protein were confirmed by RT-PCR and western blotting, respectively. At postnatal day 20, the transit period of suckling to weaning, the distribution of ERβ-positive cells was changed in the crypt-villus axis, and cytoplasm/nuclear staining changed to only nuclear staining. Moreover, Southwestern histochemistry was used to detect estrogen response element (ERE)-binding proteins, and their expression pattern was highly similar to that of ERβ. These results suggested that ERβ is the predominant ER type in mouse small intestine, and the highly similar co-localization of ERE-binding proteins reveals that ERβ is functionally active in mouse duodenum. The ERβ expression changes during postnatal development indicate that ERβ may be involved in the differentiation of duodenal epithelium.