Constitutively active estrogen receptor α (ERα) variants with C-terminal truncation are candidate molecules for gain of both endocrine- and chemotherapy-resistance in estrogen-sensitive tumors. Our previous reports using artificially truncated ERα constructs demonstrated that ERα variants encoded in 1-2-3-cryptic exon- and 1-2-3-4-cryptic exon-types of transcripts have potentials to display constitutive transactivation of an estrogen response element reporter. However, naturally occurring 1-2-3-cryptic exon-type ERα variants have not been cloned yet. Therefore, the present study was designed to identify naturally occurring ERα variants encoded in 1-2-3-cryptic exon-type ERα transcripts. We cloned a novel C-terminally truncated ERα variant (ERαi34) encoded in a 1-2-3-i34 transcript from MCF-7 cells and characterized its constitutive and ER antagonist-resistant transactivation in transfected cells. Stable transfection of the variant into MCF-7 cells augmented basal cell proliferation insensitive to fulvestrant. Collectively, we validated the structure-based mechanisms underlying constitutive and ER antagonist-resistant transactivation by C-terminally truncated ERα variants.