In the context of the separation of uranium and plutonium from spent fuel allowed by N,N-dialkylamides, three regioisomers of N,N-di(2-ethylhexyl) butyramide (DEHBA or ββ) and the diastereopure isomers of N-(2-ethylhexyl)-N-(oct-3-yl)butyramide (EHOBA or αβ) were synthesized to assess their extraction performance and to study the mechanisms at the origin of the differences observed between the stereo- and regioisomers. The N,N-dialkylamides showed differences in extraction, with a greater effect of regio- than stereoisomerism. A mechanistic study at both the molecular and supramolecular scales was initially applied to explain these effects. X-ray absorption and UV-vis spectroscopy showed that uranium is extracted by a UO2(NO3)2L2 complex, which is not very sensitive to steric hindrance, while plutonium is extracted by two complexes, Pu(NO3)4L2 and Pu(NO3)6(HL)2, which are differently affected by stereo- and regioisomerism. Investigations at the supramolecular scale also showed that Pu(NO3)4L2 complexes are disadvantaged by the bulkiness of the extractants, while Pu(NO3)6(HL)2 is favored by the preformation of larger supramolecular aggregates.