BACKGROUND: Sugars may act as either energy substrates or non-penetrating cryoprotectants. OBJECTIVE: Inclusion of non-penetrating trehalose was tested in extenders for the cryopreservation of Tambaqui ( Colossoma macropomum) sperm. MATERIALS AND METHODS: Sperm was extended 1/9 (v/v) in Beltsville Thawing Solution (BTS) with 10% DMSO (control) or 50, 100, 150 and 200 mM trehalose without 10% DMSO. Post-thawed sperm quality was evaluated, including fertilization and hatching rates, sperm motility, motility period and viability, integrity of sperm membrane and DNA, and mitochondrial functionality. RESULTS: Extenders with 100 ∼ 150 mM trehalose achieved fertilization and hatching rates similar to those of the 10% DMSO-treated sperm samples. Trehalose at 100 and 150 mM provides better protection than 10% DMSO treatment for sperm motility, viability, DNA integrity and mitochondrial functionality. Fertilization and hatching rates were highly correlated (r = 0.95, P < 0.001). CONCLUSION: The addition of 100 ∼ 150 mM trehalose in extender can replace 10% DMSO for the cryopreservation of C. macropomum sperm.