The vagina is a long muscular organ that extends from the vulva to the cervix. The vagina can elongate to accommodate the stallion penis, an inseminator's arm, or the birth of a foal. The vagina is separated by a fold of tissue (transurethral fold) located at the vulvo-vaginal sphincter. The part nearest the rear of the animal is the vestibule (posterior vagina), with anterior vagina being closer to the cervix. The vulvo-vaginal sphincter and associated fold of tissue covers the urethral opening (where the bladder empties), preventing urine flow back toward the cervix. The transurethral fold in a virgin filly is covered with a thin layer of connective tissue similar to the hymen in women. Care needs to be taken when entering the vagina of a virgin filly so as not to tear the rest of the tissue. The vestibule contains vestibular glands which secretes mucous to lubricate the posterior tract.

At the tip of the anterior vagina is the cervix. The blind pocket in the anterior vagina that surrounds the protrusion of the cervix is called the fornix.