Since it is reported to be difficult to establish Asiatic golden cat (Catopuma temmincki) breeding pairs in captivity as a result of overaggressive behavior of the male, artificial insemination (AI) may be a desired option by which to achieve pregnancy. This approach was chosen in the present case involving a nulliparous, 6-yr-old female cat that was inseminated transcervically during a naturally occurring estrus, which therefore required only a single general anesthetic procedure. On day 4 of estrus behavior, the male was anesthetized and semen was collected via urethral catheterization (UC) to recover spermatozoa in high concentration followed by electro-ejaculation (EE) to obtain additional semen and seminal fluid. The fresh UC semen, totaling 180 μl in volume and containing spermatozoa showing 55–70% sperm motility, was inseminated 2.5 hr later via a commercial cat urinary catheter passed through the cervix into the uterus. Immediately afterwards, the EE fraction (100 μl) was inseminated deeply into the dorsal medial fold of the vagina. The GnRH analogue Receptal® (0.75 ml, i.m.) was given during anesthesia in an attempt to induce ovulation. Increasing fecal concentrations of progesterone after AI and a significant rise in fecal prostaglandin F2α metabolite (PGFM) concentrations (P < 0.0001) from day 45 post-AI indicated that the cat had conceived, and it produced healthy twin cubs after an 84-day gestation.