ABSTRACTMetabolism associated with energy production is highly compartmentalized in eukaryotic cells. During this process, transporters that move metabolites across organelle membranes play pivotal roles. The highly conserved ADP/ATP carrier (AAC) involved in ATP and ADP exchange between the mitochondria and cytoplasm is key to linking the metabolic activities in these 2 compartments. The ATP produced in mitochondria can be exchanged with cytoplasmic ADP by AAC, thus satisfying the energy needs in the cytoplasm.Toxoplasma gondiiis an obligate intracellular parasite with a wide range of hosts. Previous studies have shown that mitochondrial metabolism helpsToxoplasmato parasitize diverse host cells. Here, we identified 2 putative mitochondria ADP/ATP carriers inToxoplasmawith significant sequence similarity to known AACs from other eukaryotes. We examined the ATP transport function ofTgAACs by expressing them inEscherichia colicells and found that onlyTgAAC1 had ATP transport activity. Moreover, knockdown ofTgAAC1 caused severe growth defects of parasites and heterologous expression of mouse ANT2 in theTgAAC1 depletion mutant restored its growth, revealing its importance for parasite growth. These results verified thatTgAAC1 functions as the mitochondrial ADP/ATP carrier inT. gondiiand the functional studies demonstrated the importance ofTgAAC1 for tachyzoites growth.IMPORTANCET. gondiihas an efficient and flexible energy metabolism system to meet different growth needs. ATP is an energy-carrying molecule and needs to be exchanged between organelles with the assistance of transporters. However, the function ofTgAACs has yet to be characterized. Here, we identified 2 putative AACs ofT. gondiiand verified that onlyTgAAC1 had ATP transport activity with expression in the intactE. colicells. Detailed analyses found thatTgAAC1 is critical for the growth of tachyzoites andTgAAC2 is dispensable. Moreover, complementation with mouse ANT2 restored the growth speed of iTgAAC1, further suggestingTgAAC1 functions as a mitochondrial ADP/ATP carrier. Our research demonstrated the importance ofTgAAC1 for tachyzoites growth.