In this study, we have examined the influence of different lipids on the solubility of the xanthophyll cycle pigments diadinoxanthin (Ddx) and violaxanthin (Vx) and on the efficiency of Ddx and Vx de-epoxidation by the enzymes Vx de-epoxidase (VDE) from wheat and Ddx de-epoxidase (DDE) from the diatom Cyclotella meneghiniana, respectively. Our results show that the lipids MGDG and PE are able to solubilize both xanthophyll cycle pigments in an aqueous medium. Substrate solubilization is essential for de-epoxidase activity, because in the absence of MGDG or PE Ddx and Vx are present in an aggregated form, with limited accessibility for DDE and VDE. Our results also show that the hexagonal structure-forming lipids MGDG and PE are able to solubilize Ddx and Vx at much lower lipid concentrations than bilayer-forming lipids DGDG and PC. We furthermore found that, in the presence of MGDG or PE, Ddx is much more solubilizable than Vx. This substantial difference in Ddx and Vx solubility directly affects the respective de-epoxidation reactions. Ddx de-epoxidation by the diatom DDE is saturated at much lower MGDG or PE concentrations than Vx de-epoxidation by the higher-plant VDE. Another important result of our study is that bilayer-forming lipids DGDG and PC are not able to induce efficient xanthophyll de-epoxidation. Even in the presence of high concentrations of DGDG or PC, where Ddx and Vx are completely solubilized, a strongly inhibited Ddx de-epoxidation is observed, while Vx de-epoxidation by VDE is completely absent. This indicates that the inverted hexagonal phase domains provided by lipid MGDG or PE are essential for de-epoxidase activity. We conclude that in the natural thylakoid membrane MGDG serves to solubilize the xanthophyll cycle pigments and furthermore provides inverted hexagonal structures associated with the membrane bilayer, which are essential for efficient xanthophyll de-epoxidase activity.