We studied the Persian onager (Equus hemionus onager), an endangered equid subspecies. The objective was to characterize endocrine patterns and ovarian follicular dynamics of females as well as seminal traits and sperm sensitivity to cryopreservation in males as a prerequisite to testing the feasibility of artificial insemination (AI). Urinary progesterone and estrogen metabolite profiles were determined by enzyme immunoassay in 11 females. Serial ultrasonography of ovarian activity was performed for 2 mo in a subset of four females. Females were seasonally polyestrous (June–November). Ovarian morphometry via ultrasonography and urinary progesterone profiles were more reflective of reproductive events than urinary estrogen patterns, and preovulatory follicle size was smaller than reported for other equid species. There was evidence for lactational suppression of estrus for up to 1.5 yr in nursing dams. Electroejaculation allowed recovery of highly motile sperm from 7, anesthetized males on 57% of occasions. Spermatozoa, including motility and acrosomal integrity, were resilient to freeze-thawing. Artificial insemination was successful in 2 of 3 females following detection of a dominant follicle and deslorelin administration, resulting in births of a healthy female and male foal by using fresh/chilled and frozen/thawed sperm, respectively.