Abstract The role of the estrogen receptor β (ERβ) in the colon has received considerable interest, yet in vivo models are needed to better define its protective actions. In the present study, wild-type (WT), ERα, and ERβ knockout (αERKO and βERKO) mice were injected with azoxymethane, a colon chemical carcinogen. Fourteen weeks after azoxymethane exposure, the incidence of aberrant crypt foci (ACF) was assessed by methylene blue staining. βERKO mice showed significantly higher incidence (P < 0.001) of ACF (15.0 ± 2.5) compared with αERKO (3.4 ± 1.0) and WT (4.6 ± 1.0) mice. The colons in several βERKO mice had increased thickness and loss of normal morphology. It has been reported that ERβ plays a role in the maintenance of the colonic crypt architecture; this may explain the loss of crypt organization in the colonic epithelium of βERKO mice. The presence of mucin-depleted foci (MDF) has been shown, both in humans and in rodents, as an early event in colon cancer. Therefore, to surpass the limitations with ACF scoring, we performed Alcian blue-neutral red staining to assess the presence of MDF. This assay allowed the assessment of precancerous lesions on all the βERKO mice colons (38.3 ± 4.0; P < 0.001), comparing to WT and αERKO mice (6.6 ± 1.5 and 10.0 ± 1.9, respectively), and served to confirm the ACF results. Together, these data support the use of MDF staining as a biomarker for precancerous lesions and the protective role of ERβ in colon carcinogenesis. Cancer Prev Res; 3(9); 1198–204. ©2010 AACR.