To date, no structural study has been carried out on the effects of vitamin A deficiency (VAD) on hippocampal macromolecules. Therefore, in the present study, the effect of dietary VAD on the structure, content and function of rat hippocampal molecules was investigated using Fourier transform IF spectroscopy. Male Wistar rats were randomly divided into three groups: an experimental group maintained on a vitamin A-deficient liquid diet (VAD, n 7); a control group maintained on a vitamin A-supplemented liquid diet (CON, n 9); a pure control group maintained on standard solid laboratory chow (PC, n 7). The PC group was included in the study to ensure that the usage of liquid diet did not influence the outcomes of VAD. Both the CON and PC groups were successfully discriminated from the VAD group by principal component analysis and hierarchical cluster analysis. The spectral analysis indicated a significant decrease in the contents of saturated and unsaturated lipids, cholesteryl esters. TAG and nucleic acids in the VAD group when compared with the CON group (P <= 0.05). In addition, a significant decrease in membrane fluidity and a significant increase in lipid order (e.g. acyl chain flexibility) were observed in the VAD group (P<0.001). The results of the artificial neural network analysis revealed a significant decrease in the alpha-helix structure content and a significant increase in the turn and random coil structure contents, indicating protein denaturation, in the VAD group when compared with the CON and PC groups (P <= 0.05). Dietary exclusion of vitamin A for 3 months apparently had an adverse impact on compositional, structural and dynamical parameters. These changes can be due to increased oxidative stress, confirming the antioxidant protection provided by vitamin A when used as a dietary supplement at low-to-moderate doses.