The aim of this study was to analyze the effect of reduced glutathione (GR) and adenosine triphosphate (ATP) in cryopreserved sperm tambaqui, Colossoma macropomum..Semen samples were frozen at different concentrations of GR (2, 4, and 6 mM) and ATP (7.5, 15, 30 mM) evaluated separately.Different concentrations of GR and ATP were added to the diluent Beltsville Thawing Solution 10 % of DMSO (extender control). The thawed sperm was evaluated as the kinetic parameters for Computer Assisted Semen Analysis (CASA) and cell feature parameters by flow cytometry.All concentrations of GR and ATP had gradual reduction in the production of reactive oxygen species, mainly ATP 15; 22.5 mM and 30 mM, which decreased by 50% or more when compared to the control. All concentrations of GR and ATP did not change (P> 0.05) DNA fragmentation and lipid peroxidation. The membrane fluidity GR in concentrations 4 and 6 mM were better than control; and the concentrations of 2 and 4 mM maintained more efficiently to motility period, total and progressive motility.Thus, it is indicated the use of BTS extender with 10% DMSO added 4 mM GR thawed sperm Tambaqui C. macropomum.