The teleost matrinxã Brycon orthotaenia, native to the São Francisco River in Brazil, is in the vulnerable category of species in threat of extinction in the State of Minas Gerais, Brazil. We developed a protocol to freeze its spermatozoa to be used in artificial breeding programs aimed at preserving this species. Hand-stripped milt measured 2.0 ± 0.8 ml with a sperm concentration of 14.4 x 109 spermatozoa/ml, and a spermatocrit of 40.2 ± 9.6%. We used post-thaw sperm motility to evaluate the effects of the freezing protocol on the spermatozoa. To measure sperm motility of fresh samples, we used a two-step dilution procedure. Fresh spermatozoa remained immotile when the milt was diluted in a 150- or 200-mM NaCl solution, but motility rates rose when the milt was diluted in NaCl solutions of lower osmolality. Peak motility occurred in a 25- and 50-mM NaCl solutions (88% motile spermatozoa and >50 s duration of motility). Concentrations of 5%, 10%, and 15% of DMSO, ethylene glycol, and propanediol did not significantly induce changes in motility rate of fresh spermatozoa. However, exposure to methanol and dimethyl acetamid in the same concentrations resulted in significantly lower motility rates. We performed cryopreservation assays in a dry shipper cryogenic container. Milt was frozen in extender solutions (1:5 dilution, v/v) containing 10% DMSO, 10% propanediol, or 10% ethylene glycol plus 5% glucose, 10% hen egg yolk, and 75% distilled water. We obtained the highest post-thaw motility rate and duration of sperm motility (70.5 ± 11.4% and 62.2 ± 8.5 s respectively) when DMSO was the cryoprotectant and the thawed spermatozoa were activated with 119 mM NaHCO3. Thus, the cryopreservation protocol developed in this work successfully preserved spermatozoa of matrinxã.