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Simple Summary Cryopreserving dog semen allows the long-term availability of male gametes for future artificial insemination and other assisted reproductive techniques. However, freezing causes irreversible damage to sperm that can affect its ability to fertilize and generate a viable pregnancy. Sperm alterations are partly attributed to oxidation produced by reactive oxygen species (ROS); therefore, antioxidants have been included as extenders for seminal cryopreservation. The unconventional natural antioxidants might reduce deleterious changes in cryopreserved dog sperm; therefore, we evaluated the effects of cryopreservation with the antioxidants ergothioneine and isoespintanol on thawed canine sperm. Various concentrations of both antioxidants improved the movement capacity and structure of thawed spermatozoa, possibly by reducing ROS production. The unconventional antioxidants isoespintanol and ergothioneine improved the quality of cryopreserved canine semen and hence improved assisted canine reproduction. Abstract Sperm undergo oxidative stress due to excessive production of reactive oxygen species (ROS) during cryopreservation. Some unconventional natural antioxidants can reduce ROS-induced changes in cryopreserved canine sperm. This study aimed to identify the cryoprotective effects of ergothioneine and isoespintanol on the quality of thawed canine semen. Twelve ejaculates from six dogs were cryopreserved in a tris-yolk extender without (control) or with 50 (E50), 100 (E100), or 150 (E150) µM ergothioneine or 20 (I20), 40 (I40), or 60 (I60) µM isoespintanol. We evaluated the motility and kinetics of thawed sperm using computerized analysis; determined morphology by eosin-nigrosin staining; functional membrane integrity using hypoosmotic tests, and structural membrane and acrosome integrity; mitochondrial membrane potential by fluorescence microscopy; and ROS production by spectrophotometry. Data were statistically analyzed using mixed models and Tukey tests. E100 increased total (60.6% vs. 49.6%) and progressive (26.4% vs. 20.1%) motility, straight line velocity (41.3 vs. 35.9 µm/s), and rapid sperm (17.6% vs. 12.3%) compared with controls. However, E150 reduced the numbers of hyperactive sperm. E100, I40, and I60 reduced the abnormal morphology and ROS production, and all concentrations of both antioxidants increased acrosomal integrity. We concluded that ergothioneine and isoespintanol reduce deleterious sperm alterations and oxidative stress in thawed canine semen.