Sperm cryopreservation, in combination with assisted reproductive techniques, is a valuable tool for the genetic management of endangered felids. However, the acrosome of the cheetah spermatozoon is especially sensitive to cryopreservation, with 40% of spermatozoa experiencing acrosomal damage immediately after thawing and then another 15% loss during the next 4 h in vitro. Additionally, thawing causes a reduction in sperm motility by 20% with another decrease of 12% during subsequent incubation in vitro. We hypothesized that slow removal of glycerol from cryopreserved cheetah spermatozoa using an Accudenz gradient would improve acrosomal integrity, sperm motility longevity and structural morphology. Accudenz was compared to traditional cheetah sperm processing methods for glycerol removal that involved washing, multi-step resuspension and swim-up processing. Electroejaculates (n = 21; n = 8 males) were washed in Ham's F10 medium (HF10) and sperm pellets resuspended in TEST Yolk-Buffer (TYB) with 0% glycerol. Samples were cryopreserved in straws in 4% final glycerol, thawed and assessed for percent intact acrosomes (% IA), percent motility (% M) and forward progressive status (FPS; scale 0-5). Sperm motility index (SMI) was calculated as [% M + (FPS x 20)] ÷ 2. In Study 1, glycerol removal by centrifugation through an Accudenz gradient (4%, 10%) was compared to traditional sperm washing (control) and multi-step resuspension protocols. At each time after centrifugation (hourly for 4 h), % IA was improved (P < 0.05) for Accudenz (range, 36 - 39%) compared to control (30 - 33%) and multi-step (29 - 33%) treatments. In Study 2, a modified Accudenz protocol was compared to traditional washing and found to improve (P < 0.05) SMI (range, 52 - 64) compared to controls (range, 41 - 52) at each time post-thaw after centrifugation. In Study 3, swim-up processed sperm were compared to centrifugation through Accudenz and traditional sperm washing for improving sperm morphology. Percentage of structurally-normal sperm recovered post-thawing increased (P < 0.05) for both the Accudenz (38%) and swim-up (33%) treatments compared to controls (21%). Percent IA and SMI also were improved (P < 0.05) for Accudenz (range, 39 - 47%; 46 - 59, respectively) compared to controls (range, 26 - 33%; 40 – 53, respectively). Results indicate that using Accudenz for glycerol removal from cryopreserved cheetah sperm mitigates the significant loss in sperm quality that occurs after freeze-thawing. This alleviation of cellular damage resulting from cryopreservation contributes to a >10% improvement in overall sperm motility and, more importantly, allows retaining 40% or more of sperm with intact acrosomes.