The objective of this study was to examine the interaction of different concentrations of trehalose [0 (T0), 50 (T50) or 100 (T100) mM] and glycerol [5% (G5) or 7% (G7)] on post-thawed quality of ram semen, cryopreserved in a soybean lecithin (SL)-based extender. Twenty-eight ejaculates were collected from four rams and diluted with six trehalose/glycerol combinations: T0G5, T50G5, T100G5, T0G7, T50G7, and T100G7. Sperm motility (CASA), membrane integrity (eosin/nigrosin) and functionality (HOST), abnormal forms, capacitation status (CTC), mitochondrial activity (rhodamine 123), apoptotic features (Annexin V/propidium iodide) and lipoperoxidation (malondialdehyde production) were evaluated after thawing. Extender T100G5 yielded the highest results for total and progressive motility, sperm velocity, normal morphology, functional membranes, active mitochondria and membrane integrity, with P<0.05 in general, except for T50G7 (P>0.05). The combinations T0G5, T0G7 and T100G7 yielded the lowest post-thaw quality. We could not detect significant changes in other kinematic parameters, capacitation status or lipoperoxidation. We conclude that, in our SL-based extender, a combination of 100 mM trehalose and 5% glycerol was the most adequate combination to achieving post-thawing quality in our soybean lecithin-based extender, and our results support that a synergistic effect among trehalose and glycerol exists. We suggest that other combinations could improve these results.