Microstructural evolution of a cationic gemini surfactant butanediyl-1,4-bis(dodecyldimethylammonium bromide) (hereafter referred to as 12-4-12) micelles in the presence of two bile salts viz. sodium deoxycholate (NaDC) and sodium cholate (NaC) was investigated using surface tension, viscosity, nuclear magnetic resonance (NMR), and small angle neutron scattering (SANS) measurements. A negative value of interaction parameter (β) evaluated from surface tension measurements is a signature of strong synergistic interaction between oppositely charged surfactants. Other micellar parameters have also been calculated at different mole fractions. Both the bile salts induced a shape transitions in 12-4-12 micelles on account of their hydrophobicity. Viscosity measurements disclose that loading of bile salts induces morphological changes in gemini micelles; NaDC is more efficient in altering the aggregation behaviour compared to NaC and presents a pronounced increase in viscosity and micellar growth which is suppressed at elevated temperatures. A remarkable growth observed in micelles in the presence of NaDC at low pH has been ascribed to the solubilization of bile acids formed in acidic medium. The size and shape of gemini mixed micelles obtained from SANS measurements are explicated on the basis of the hydrophobicity of bile salts. The location of bile salts in the micelle was determined from NOESY. The present study characterizes gemini–bile salt mixed systems which significantly enriches our knowledge and such a structural transition provides an opportunity to use these bioamphiphiles (bile salt containing mixed micelles) as delivery vehicles and in some pharmaceutical formulations.