We report the molecular cloning of a H⁺-ATPase in the symbiotic dinoflagellate,Symbiodiniumsp. previously suggested by pharmacological studies to be involved in carbon-concentrating mechanism used by zooxanthellae when they are in symbiosis with corals. This gene encodes a protein of 975 amino acids with a calculated mass of about 105 kDa. The structure of the protein shows a typical P-type H⁺-ATPase structure (type IIIa plasma membrane H⁺-ATPases) and phylogenetic analyses show that this new proton pump groups with diatoms in the Chromoalveolates group. ThisSymbiodiniumH⁺-ATPase is specifically expressed when zooxanthellae are engaged in a symbiotic relationship with the coral partner but not in free-living dinoflagellates. This proton pump, therefore, could be involved in the acidification of the perisymbiotic space leading to bicarbonate dehydration by carbonic anhydrase activity in order to supply inorganic carbon for photosynthesis as suggested by earlier studies. To our knowledge, this work provides the first example of a symbiosis-dependent gene in zooxanthellae and confirms the importance of H⁺-ATPase in coral–dinoflagellate symbiosis.