Ga2O3 is an ultra-wide-bandgap semiconductor that is receiving considerable attention due to its promising applications in high-frequency, high-power and high-temperature settings. It can be prepared by calcinating the α-GaOOH phase at high temperatures. Understanding the significance of hydroxyl groups in α-GaOOH, dehydroxylation and the structural transition at high temperatures has become a key aspect of preparing high-quality α-Ga2O3 crystals, but the underlying mechanism remains unknown. In this research, α-GaOOH nanorods were hydrothermally synthesized and the structural evolution of α-GaOOH investigated at high temperatures by in situ X-ray diffraction. The hydroxyl group in α-GaOOH squeezes Ga3+ from the center of the [GaO6] octahedron, resulting in deformed [GaO6] octahedra and significant microstrain in α-GaOOH. The hydroxyl groups are peeled off from α-GaOOH when the temperature exceeds 200°C, resulting in contraction along the c-axis direction and expansion along the a-axis direction of α-GaOOH. When the temperature exceeds 300°C, the Ga—O bond inside the double chains preferentially breaks to generate square-wave-like octahedron chains, and the neighboring chains repack to form hexagonal-like octahedron layers. The octahedron layers are packed up and down by electrostatic interaction to generate the α-Ga2O3 structure. This work highlights the role of hydroxyl groups in α-GaOOH, dehydroxylation and the structural transition on the atomic scale, providing valuable guidelines for the fabrication of high-quality α-Ga2O3 crystals.