It has been established that a minute difference of the primary chemical and topological structures influences the self-assembly behaviors of giant surfactants; however, the regio-configuration effect has been rarely reported. Herein, we report a systematic study on the self-assembly behaviors of a series of double-chain giant surfactant regio-isomers, which consist of a hydrophilic polyhedral oligomeric silsesquioxane (POSS) head and two identical hydrophobic polystyrene (PS) tails with various molecular weights tethered in para-, meta-, and ortho-configurations, respectively. Small-angle X-ray scattering and transmission electron microscopy characterizations have been combined to investigate the phase behaviors of each sample and construct the phase diagrams for the three isomers with respect to the molecular weights of PS tails. It is observed that the regio-configuration significantly impacts the self-assembly behaviors of the giant surfactant isomers, including order-to-disorder (ODT) and order-to-order (OOT) transitions. In each isomer system, the order-to-disorder transition temperature (T-ODT) changes nonmonotonically with the length of PS tails, which is generally similar to the variation of T-ODT for block copolymers but also exhibits some peculiar features associated with the rigid conformation of headgroup. With equal length of PS tails, T-ODT of the three isomers is in the descending order of ortho > meta > para. There is a pronounced and systematic phase boundary shift to lower volume fraction of PS (f(PS)) and to lower temperatures from para to meta to ortho. This is closely related to the compounds regio-configuration and rigid 3D conformation of the headgroup and may be understood through the lower effective f(PS) as two tails get further apart. These findings elucidate the sophisticated effect of the regio-configuration on self-assembly behaviors. It suggests regio-configuration as an additional factor in tuning the self-assembly of giant surfactants at sub-10 nm or even sub-5 nm length scales, which is of significant technical importance.