Many marine fishes have adaptive respiratory strategies to enable sufficient oxygen (O₂) uptake, as well as the excretion of metabolically-produced carbon dioxide (CO₂). A central figure in the intertwined systems of CO₂ excretion and tissue O₂ delivery is the metalloenzyme carbonic anhydrase (CA). In my dissertation I expand, and in some cases challenge, the classical role of CA in CO₂ excretion and its emerging role in tissue O₂ extraction in both the red blood cell (RBC) and tissue membranes. I investigated the role of RBC CA in a diverse group of fishes, and demonstrated basal fishes with a membrane-bound CA isoform in their gills (branchial CA-IV) possess a low-activity RBC CA. Using site-directed mutagenesis, I restored increased function to a basal fish RBC CA to demonstrate that a single amino acid is critical for CA function. Furthermore, phylogenetic analysis suggested high-activity RBC CA may have coevolved with enhanced hemoglobin (Hb) pH sensitivity in teleosts. I explored this relationship and demonstrated RBC CA activity dictated Hb-O₂ offloading rate in almost a 1:1 manner in red drum (Sciaenops ocellatus). RBC CA is best known for its role in CO₂ excretion so this study is the first to demonstrate RBC CA may be rate-limiting for O₂ offloading as well. An additional CA isoform has recently been implicated in tissue O₂ extraction: membrane-bound CA-IV found in the red muscle, heart, and eye. With the plethora of CA-IV isoforms that function in other physiological systems (e.g. acid-base and ion regulation), I sought to define which isoforms should be studied for respiration, using publicly available membrane-bound CA sequences for a comprehensive phylogeny, and paired with tissue distribution analyses. I demonstrated functional divergence in CA-IV isoforms in which some species possess multiple CA-IV isoforms for disparate physiological functions. This highlighted CA-IVa as the primary isoform to target for future respiratory gas exchange studies. Finally, I challenged fish with low O₂ exposure (hypoxia) to understand the role CA-IV may play under environmental stress, and in contribution to whole-animal performance. While fish did not recruit CA-IV under hypoxia acclimation as predicted, they maintained CA-IV protein synthesis to theoretically sustain tissue O₂ extraction. Furthermore, hypoxia acclimation improved swim performance under control conditions; however, anaerobic rather than aerobic processes seem to be driving this performance. Overall, my work presents critical information regarding the emerging roles of CA in tissue O₂ extraction in marine fishes, providing mechanistic and evolutionary insight on the enzyme’s function in respiratory gas exchange.