Heteromeric amino acid transporters (HATs) are composed of a light chain from the SLC7 family and a heavy chain from the SLC3 family, forming obligate heterodimeric complexes that mediate the transport of amino acids, thyroid hormones, and amino acid–like drugs across cellular membranes. These transporters are essential for maintaining nutrient homeostasis and regulating cell growth and signaling. Mutations in HAT components are associated with several inherited disorders, including cystinuria, lysinuric protein intolerance, and certain forms of autism spectrum disorder. Despite their physiological and pathological significance, the molecular mechanisms underlying HAT assembly, substrate recognition, and transport dynamics remain poorly understood. Here, we employed cryo-electron microscopy (cryo-EM) in combination with biochemical and functional analyses to resolve multiple conformational states of HATs. The resulting structures reveal key molecular features that define the assembly interface between the light and heavy chains and capture conformational transitions associated with substrate translocation. These findings provide deep mechanistic insights into HAT function and establish a structural framework for the rational design of selective therapeutics targeting this important transporter family.