Optimizing cryopreservation protocols for nondomestic felids contributes to the successful development of assisted reproduction techniques and genetic resource banking. In this study, we describe a simple cryopreservation procedure for African lion (Panthera leo) ejaculates, which was tested with different packaging options and different sperm numbers per dose. By applying urethral catheterization and electroejaculation, 17 ejaculates with greater than 20% motile and greater than 5% progressively motile sperm were collected. A lyophilized extender (a modified egg yolk-Tes-Tris-fructose-glycerol medium) was rehydrated and added in one step at ambient temperature (∼25 °C) to semen, which was prediluted in cell culture medium M199. After slow cooling of insulated samples to 15 °C in a refrigerator (4 °C), the samples were fast frozen over the surface of liquid nitrogen or in a dry shipper. Aliquots of 300 μL containing 20 × 106 sperm were frozen in cryovials and in 0.5-mL straws. Differences were observed in the total motility after thawing between vial (31.5 ± 14.1%) and straw freezing (20.1 ± 8.6%). However, the subpopulations of vital (22.7 ± 7.8% for vial and 19.8 ± 8.5% for straw) and progressively motile (10.0 ± 7.9% for vial and 10.0 ± 6.4% for straw) sperm after washing and 1 hour incubation at 38 °C were of similar magnitude, velocity, and linearity for both packaging options. After freezing of five ejaculates with 20, 60, and 100 × 106 sperm per dose, best results were achieved at the lowest concentration. In general, post-thaw results were highly variable (2.2% and 56.5% total motility) and not correlated to motility or morphology of the fresh semen. To further characterize semen quality, we assessed the protective potential of seminal fluid against oxidative stress, which might be challenged on freeze thawing. The capacity of seminal fluid to reduce radicals was measured in 10 semen samples by electron spin resonance spectroscopy and a spin-labeled fatty acid as a radical probe. Moreover, we determined the lysophosphatidylcholines (LPC) as potential lipid oxidation products in the sperm and erythrocytes of the males. Individuals with a high radical reduction capacity in the seminal fluid and a low LPC content in their erythrocytes showed a better cryosurvival of sperm. This is a first indication that seminal fluid may affect the freezing potential of African lion ejaculates.