Amino acid transporters are essential for maintaining intracellular and extracellular amino acid homeostasis. The sodium-dependent neutral amino acid transporter B⁰AT1 (SLC6A19) requires the accessory protein ACE2 or Collectrin to form a functional heteromeric complex for transmembrane transport. B⁰AT1 is primarily expressed at the brush border of epithelial cells in the small intestine and kidney, playing a vital role in the absorption and reabsorption of neutral amino acids, including Leucine (Leu), Methionine (Met), Glutamine (Gln), Tryptophan (Trp) and Phenylalanine (Phe). Mutations or functional impairments in B⁰AT1 lead to Hartnup disorder, characterized by aminoaciduria and neurological symptoms. Although its physiological roles are increasingly understood, the molecular mechanisms underlying selective substrate recognition, particularly for aromatic amino acids, remain poorly understood. Here, we report high-resolution cryo-electron microscopy (cryo-EM) structures of the human ACE2-B⁰AT1 complex bound to Phe and Trp at the overall resolution of 2.87 Å and 3.24 Å, respectively. Structural comparisons reveal conserved substrate backbone anchoring and distinct side-chain recognition mechanisms, identifying key residues that modulate substrate specificity. Our findings elucidate the substrate recognition landscape of B⁰AT1 and provide a mechanistic framework for understanding its function and disease-associated mutations.