Vitamin D plays an important role in many physiological processes, particularly calcium and phosphorous homeostasis. The biochemistry of vitamin D is also complex, encompassing a range of active molecules that may be either endogenous or dietary in origin. The role of lipids and fats in the production, processing and use of vitamin D is an interesting one, with a relative paucity of model studies into the interactions of vitamin D with lipidic systems such as micelles and vesicles. Here, we have studied the effect of vitamin D3 in simple unsaturated phospholipid systems. We used NMR and FTIR spectroscopy to investigate the effect of increasing vitamin D concentration on the structure and dynamics of the lipid chains and interfacial region. In order to link these model studies with more complex biomimetic environments, we compare results in the presence of buffer and vitamin D binding protein. We have also used DLS to determine that vitamin D3-DOPC vesicles can retain their size distribution for varying amounts of time in different conditions. We find that the acyl chain region of vitamin D3-DOPC membranes are generally disordered, and that the addition of buffer and/or protein alters the properties of the interfacial region.