Our results provide mechanistic insight into IGF/IGFBP3/ALS ternary complex assembly and its disassembly upon proteolysis for IGF bioavailability, suggesting a structural basis for human diseases associated with IGF1 and IGFALS gene mutations such as complete ALS deficiency (ACLSD) and IGF1 deficiency. Here, IGFBP3 is linked to hyperinsulinemic hypoglycemia, familial, 4.