Cuniculus paca is a terrestrial rodent that inhabits the neotropical forests of Central and South America. The aim of this study was to evaluate the effect of sperm separation on the quality of cryopreserved semen of C. paca. Sixty straws of frozen semen were thawed. One aliquot was subjected to sperm separation by single-layer density gradient centrifugation (SLC) and the other aliquot remained as a control group with semen not separated. Sperm motility and kinetics were evaluated using a computerized-assisted sperm analysis (CASA) system, functional membrane integrity by hypoosmotic test (HOST), and sperm morphology by eosin-nigrosin staining. The adjustment of linear models and comparison of means by Tukey was performed. The SLC separation produced a higher proportion of motile and linear spermatozoa, while the control showed higher values for curvilinear velocity (p˂0.05). Likewise,...