Ovarian steroid hormones, estrogens, are now regarded as multifunctional hormones, and have pivotal roles in modulation of neuroendocrine functions. The actions of estrogens are mediated via two subtypes of nuclear estrogen receptors, estrogen receptor α and β (ERα and ERβ, encoded separately in ESR1 and ESR2, respectively). The structure of the human ERβ gene was first reported in 1996, and several splice variants have been described in subsequent work. However, it remains to be fully elucidated. Therefore, we decided to identify other human ERβ splice variants and to re-examine the genomic organization of the human ERβ gene. We cloned novel C-terminally-truncated variants from the testis using rapid amplification of cDNA 3’-ends, and identified a novel terminal exon and a novel alternative splice acceptor site. Subsequently, we comprehensively analyzed the distribution of human ERβ variants in the human peripheral organs and brain subregions using reverse transcription-polymerase chain reaction. The variant mRNAs were detected in a wide range of organs including the adrenal grand, spleen, and thymus, and the various regions of the brain such as the cerebellum, amygdala and hypothalamus. Moreover, we constructed expression vectors encoding wild-type and splice ERβ variants, and characterized subcellular localization and transactivation functions of the variants in transfected cells. In conclusion, the ERβ gene generate C-terminally truncated variants by alternative splicing, which showed distinct localization patterns and functions. These findings provide useful information for further investigation on estrogen related physiological and pathophysiological processes.