We studied the neurotoxic effects of β25–35 amyloid fragment (β25–35) on cerebellar granule cells and the intracellular mechanisms involved. Treatment for 3 days with peptide greatly reduced the survival of 1 day in vitro (DIV) cultures kept in 5 mM KCl but slightly modified the survival of 25 mM KCl‐cultured cerebellar granule cells. We also studied the effect of glutamate on survival of undifferentiated cerebellar granules. We report no neurotoxic effect of glutamate on 3‐DIV‐treated cultures; whereas in β25–35‐pretreated cells, a significant glutamate toxicity was observed. Treatment of 6‐DIV cells with β25–35, performed with 25 mM KCl, induced a late but significant neurotoxic effect after 5 days of exposure, and death occurred within 8 days. Differentiated cerebellar granule cells were also sensitive to glutamate‐related neurotoxicity, and this effect was enhanced by β25–35 pretreatment. To study the molecular mechanisms underlying the neurotoxic effects of β25–35, changes in calcium homeostasis after glutamate stimulation were evaluated in control and β25–35‐treated cells. β25–35 did not affect basal [Ca2+]i but modified glutamate‐induced [Ca2+]i increase, causing a sustained plateau phase that persisted even after the removal of the agonist. These results show that β25–35 induces neurotoxicity in cerebellar granule cells and that this effect is related to modifications in the control of calcium homeostasis.