The oligomers of Aβ1-40 peptide in PBS buffer solution were analyzed by SEC and native PAGE, and the trimer of Aβ1-40 was also isolated by SEC. In addition, the effects of the soluble Aβ1-40 trimer on intracellular free calcium (Ca2+) balance of hippocampal neurons of postnatal rats were investigated by fluorescence microscopy. The experimental results indicated that Aβ1-40 peptide existed in the form of low molecular weight oligomers in 0.231 mmol/L fresh Aβ1-40 solution (20 mmol/L sodium phosphate buffer, pH 7.4, 0.02% sodium azide) within 24 h and the soluble trimer was the most abundant species. Both the trimeric and the fibrillar Aβ1-40 were able to increase the intracellular Ca2+ concentration, but the Aβ1-40 trimer caused a gradual rise and the potential was also stronger than that of the fibrils at the same concentration. In addition there were different response modes for trimeric and fibrillar Aβ1-40, meaning that there are different mechanisms of increase in intracellular Ca2+ caused by Aβ1-40.