Several lines of controversial evidence concerning estrogen receptor β (ERβ) remain to be solved because of the unavailability of specific antibodies against ERβ. The recent validation analysis identified a monoclonal antibody (PPZ0506) with sufficient specificity against human ERβ. However, the specificity and cross-reactivity of PPZ0506 antibody against ERβ proteins from laboratory animals have not been confirmed. In the present study, we aimed to validate the applicability of PPZ0506 to rodent studies. The antibody exhibited specific cross-reactivity against mouse and rat ERβ proteins in immunoblot and immunocytochemical experiments using transfected cells. In immunohistochemistry for rat tissue sections, PPZ0506 showed immunoreactive signals in the ovary, prostate, and brain. These immunohistochemical profiles of rat ERβ proteins in rat tissues accord well with its mRNA expression patterns. Although the antibody was reported to show the moderate signals in human testis, no immunoreactive signals were observed in rat testis. Subsequent RT-PCR analysis revealed that this species difference in ERβ expression resulted from different expression profiles related to the alternative promoter usage between humans and rats. In conclusion, we confirmed applicability of PPZ0506 for rodent ERβ studies, and our results provide a fundamental basis for further examination of ERβ functions.