Equids and primates are the only species known to express the placental hormone chorionic gonadotropin (CG). CG is a member of the heterodimeric glycoprotein family and is composed of an α subunit linked to a hormone-specific β subunit. Previously, we have reported that epidermal growth factor (EGF) regulates the equine glycoprotein hormone α subunit promoter through a protein kinase C (PKC)/mitogen-activated protein kinase (MAPK) signal transduction pathway in trophoblasts. The current study investigates the regulatory element/factors involved in the induction of equine glycoprotein α subunit gene expression by EGF. Using 5′ deletion mutagenesis, we have delineated the primary EGF/PKC responsive region of the equine α subunit gene to be located between −2039 to −2032 base pairs upstream of the transcriptional start site. The sequence within this region contains an activator protein 1 (AP-1)-like response element (TGAATCA) and is similar to a consensus AP-1 (TGAC/GTCA) response element. This element appeared to preferentially interact with a c-fos/JunD heterodimer. Stimulation by EGF induced the binding of c-fos and JunD to this element and subsequently elevated promoter activity. In conclusion, an EGF/PKC/MAPK signal transduction pathway regulates equine glycoprotein α subunit gene expression through a distinct regulatory element(s) that lies between −2039 to −2032 of the equine glycoprotein α subunit promoter in trophoblasts and involves an AP-1 complex.