We have previously reported that dietary docosahexaenoic acid (DHA) improves and/or protects against impairment of cognition ability in amyloid beta1–40 (Aβ1–40)-infused Alzheimer's disease (AD)-model rats. Here, after the administration of DHA to AD model rats for 12 weeks, the levels of Aβ1-40, cholesterol and the composition of fatty acids were investigated in the Triton X100-insoluble membrane fractions of their cerebral cortex. The effects of DHA on the in vitro formation and kinetics of fibrillation of Aβ1-40 were also investigated by thioflavin T fluorescence spectroscopy, transmission electron microscopy and fluorescence microscopy. Dietary DHA significantly decreased the levels of Aβ1-40, cholesterol and saturated fatty acids in the detergent insoluble membrane fractions of AD rats. The formation of Aβ fibrils was also attenuated by their incubation with DHA, as demonstrated by the decreased intensity of thioflavin T-derived fluorescence and by electron micrography. DHA treatment also decreased the intensity of thioflavin fluorescence in preformed-fibril Aβ peptides, demonstrating the anti-amyloidogenic effects of DHA. We then investigated the effects of DHA on the levels of oligomeric amyloid that is generated during its in vitro transformation from monomers to fibrils, by an anti-oligomer-specific antibody and non-reducing Tris-Glycine gradient (4–20%) gel electrophoresis. DHA concentration-dependently reduced the levels of oligomeric amyloid species, suggesting that dietary DHA-induced suppression of in vivo Aβ1-40 aggregation occurs through the inhibitory effect of DHA on oligomeric amyloid species.