Linkages between cold acclimation and oxidative stress in fishes are unclear and contradictory results have been published. We sought to determine whether oxidative stress occurs during cold acclimation of threespine stickleback (Gasterosteus aculeatus), and, if so, when it occurs and whether it varies among tissues. Fish were warm (20°C) or cold (8°C) acclimated for 9 weeks, and harvested during acclimation. Oxidative stress was assessed in oxidative and glycolytic muscles and liver by measuring levels of protein carbonyls and glutathione, and the activity and transcript levels of superoxide dismutase (SOD). Protein carbonyl levels increased in liver after 1 week at 8°C and then decreased after week 4, and remained unchanged in glycolytic and oxidative muscle. Glutathione levels increased in liver on day 3 of cold acclimation and may minimize oxidative stress later during acclimation. When measured at a common temperature, the activity of SOD increased in oxidative and glycolytic muscles on day 2 of cold acclimation, and on day 3 in liver, and remained elevated in all tissues compared with warm-acclimated animals. When measured at the acclimation temperature, the activity of SOD was significantly higher only at week 9 in oxidative muscle of cold-acclimated stickleback compared with warm-acclimated fish, and remained constant in glycolytic muscle and liver. Increased SOD activity in oxidative muscle may be required to prevent oxidative stress brought about by increased mitochondrial density. In both muscle and liver, SOD activity increased independently of an increase in transcript level, suggesting post-translational modifications regulate SOD activity.