A study with the aim of developing a protocol for the successful cryopreservation and thawing of African lion (Panthera leo) sperm was conducted. Semen from six chemically immobilized adult lion males was collected by electro-ejaculation and frozen using Biladyl and glycerol or dimethyl sulfoxide (DMSO) cryoprotectants at 4 and 8%. Five months after preservation in liquid nitrogen, two straws per treatment from each lion were thawed using different protocols: ambient temperature for 2 minutes, 36 °C for 30 seconds (water bath) or 50 °C for 8 seconds (water bath); and evaluated for motility and forward progression using standard procedures. The results of this trial confirmed that it is possible to freeze lion sperm using Biladyl, as a semen extender and glycerol or DMSO at 4 or 8% as cryoprotectants. There were no significant differences in post-thawed sperm motility and forward progression between the two different cryoprotectants used to cryopreserve lion sperm. However, a considerable reduction in motility (compared to fresh semen) can be expected following post freeze-thawed lion sperm using either glycerol or DMSO at 4 or 8%.