Equilibrative nucleoside transporters (ENTs) translocate hydrophilic nucleosides and nucleoside analogs across cellular membranes and are essential for determining the clinical efficacy and toxicity of nucleoside analogs. Unlike the prototypic human ENT members hENT1 and hENT2, which mediate plasma membrane nucleoside transport at pH 7.4, hENT3 is an acidic pH-activated mitochondrial and lysosomal transporter. Recent studies also demonstrate that hENT3 is involved in mitochondrial transport of nucleoside analogs and that mutation in hENT3 can result in a spectrum of genetic disorders resembling mitochondrial disorders. However, despite hENT3’s prominent role in mitochondrial toxicity, the molecular basis of hENT3-mediated transport is unknown. Therefore, we sought to examine the mechanistic basis of acidic pH-driven hENT3 nucleoside transport with site-directed mutagenesis, homology modeling, and 3H-adenosine flux measurements in mutant RNA-injected Xenopus oocytes. Scanning mutagenesis of putative residues responsible for pH-dependent transport via hENT3 revealed that the ionization states of Asp219 and Glu447, and not His, strongly determined the pH-dependent transport permissible-impermissible states of the transporter. Except for substitution with certain isosteric and polar residues, substitution of either Asp219 or Glu447 with any other residues resulted in robust activity that was pH independent. Dual substitution of Asp219 and Glu447 to Ala sustained pH-independent activity over a broad range of physiological pH (pH 5.5-7.4), which also maintained stringent substrate selectivity toward endogenous nucleosides and clinically used nucleoside drugs. Albeit the transportability of nucleoside analogs remained less affected, surprisingly, the Asp219Ala-Glu447Ala double mutant escaped inhibition by nucleoside analogs e.g. AZT, DDI and Gem in pH 7.4. Our results suggest a putative pH-sensing role for Asp219 and Glu447 in hENT3 and that the size, ionization state, or electronegative polarity at these positions is crucial for obligate acidic pH-dependent activity. Furthermore, the identification of amino acid residues in hENT3 important in intracellular nucleoside transport and the lack of inhibition of Asp219-Glu447 double mutant to anti-HIV dideoxynucleosides at pH 7.4 provide useful information for rational development of novel nucleoside analogs to potentially overcome clinical mitochondrial toxicities.