A successful spawning migration in salmon depends on their athletic ability, and thus on efficient cardiovascular oxygen (O) transport. Most teleost fishes have highly pH-sensitive haemoglobins (Hb) that can release large amounts of O when the blood is acidified at the tissues. We hypothesized that plasma-accessible carbonic anhydrase (paCA; the enzyme that catalyses proton production from CO) is required to acidify the blood at the tissues and promote tissue O extraction. Previous studies have reported an elevated tissue O extraction in hypoxia-acclimated teleosts that may also be facilitated by paCA. Thus, to create experimental contrasts in tissue O extraction, Atlantic salmon were acclimated to normoxia or hypoxia (40% air saturation for more than six weeks), and the role of paCA in enhancing tissue O extraction was tested by inhibiting paCA at rest and during submaximal exercise. Our results show that: (i) in both acclimation groups, the inhibition of paCA increased cardiac output by one-third, indicating a role of paCA in promoting tissue O extraction during exercise, recovery and at rest; (ii) the recruitment of paCA was plastic and increased following hypoxic acclimation; and (iii) maximal exercise performance in salmon, and thus a successful spawning migration, may not be possible without paCA.