The protozoan parasite Toxoplasma gondii relies on its host for essential metabolites, including purines, which it cannot synthesize de novo. This study investigates the roles of equilibrative nucleoside transporters (TgENTs), specifically TgAT1, TgENT1, and TgENT3, across different developmental stages of T. gondii. Utilizing protein-protein BLAST and position-specific iterated BLAST, we identified six genes in the T. gondii genome with high sequence similarity to human and Plasmodium falciparum ENTs, focusing on TgENT1, TgENT3, and TgAT1 due to their expression in tachyzoites and homology to the nucleoside domain. Our results demonstrate that TgENT3 plays a crucial role during chronic infection, as TgENT3 deletion reduced tissue cysts by 50%. The double knockout of TgAT1 and TgENT3 (ΔTgAT1ΔTgENT3) failed to differentiate into bradyzoites under alkaline stress, indicating that both transporters are important for differentiation. Upregulation of TgENT1 transcripts in ΔTgAT1ΔTgENT3 parasites suggests a compensatory mechanism among TgENTs. Conditional deletion of TgENT1 revealed its critical role in parasite growth and replication, with auxin-treated parasites showing reduced replication and long-term growth defects. Localization studies using immunofluorescence assays confirmed TgENT1’s association with the plant-like vacuole (PLVAC) membrane, distinguishing it from other ENTs typically found at the plasma membrane. These findings highlight the distinct and overlapping functions of TgENTs in T. gondii, emphasizing their potential as therapeutic targets for toxoplasmosis. Our study contributes to the broader understanding of nucleoside transporter functions in protozoans and suggests new avenues for drug development targeting ENT function to impair parasite survival and infection.