This study is an attempt to increase our knowledge on the effect that sperm preparation techniques have on thawed spermatozoa from wild ruminants. Semen banks for wild species can be maintained and used for artificial insemination and IVF, but it is necessary to optimize spermwork. Our objective was to assess the effect of centrifugation, dilution and oxidative stress on viability of cryopreserved sperm in the Iberian red deer (Cervus elaphus hispanicus), considering the hypothesis that washing increases the susceptibility of red deer thawed spermatozoa to oxidative stress. We used epididymal samples (200 × 10⁶ sperm mL^–1) from four Iberian red deers, stored in our wildlife cryobank. One straw per male was thawed and diluted to 30 × 10⁶ mL^–1 with BGM (Hepes-based) medium, and processed in a 2 × 4 factorial design as follows. Half of each diluted sample was centrifuged (300g, 5 min), and the pellet resuspended with fresh BGM at the same concentration. The diluted and washed samples were divided again, adding: nothing (Control), 1 mm Trolox (vitamin E), 0.1 mm Fe²⁺/0.5 mm ascorbate (oxidant), or Trolox plus oxidant. Tubes were incubated at 37°C and analyzed each hour for 3 h. Membrane (YO-PRO-1: apoptotic, merocyanine 540: stability, PI: damaged) and mitochondrial status were assessed simultaneously using YO-PRO-1/Merocyanine 540/propidium iodide/Mitotracker Deep Red staining (Invitrogen) evaluated with flow cytometry. Linear mixed-effects models were used to test incubation, additives and dilution/washing effects. The proportion of viable and non-apoptotic spermatozoa decreased with time (40% ± 1 at 0 h to 23% ± 1 at 3 h; P < 0.001). However, there were no significant differences among treatments. Merocyanine-negative spermatozoa decreased slightly with time in the diluted samples (97% ± 0.3 to 87% ± 3 at 3 h; P < 0.05), but no differences were found among treatments. However, the proportion of spermatozoa with active mitochondria (in the viable and non-apoptotic population) decreased dramatically when samples were washed and only oxidant was added (84% ± 5 to 5% ± 3 at 3 h; P < 0.001), while this parameter of the remaining treatments gradually decreased (86% ± 2 to 78% ± 1 at 3 h). These results support the hypothesis that washing increases the susceptibility of red deer thawed spermatozoa to oxidative stress. Nevertheless, in the absence of oxidative stress, gentle centrifugation had no additional detrimental effect over that of direct dilution. Moreover, the antioxidant, Trolox, abolished the negative effect of the oxidant on loss of mitochondrial activity. Although we did not observe an improvement in the absence of oxidant, it is possible that antioxidants may have a positive effect in the long term. This should be explored in future studies.