The present study established a model of RyR2 knockdown cardiomyocytes and elucidated the role of RyR2 in aconitine-induced arrhythmia. Cardiomyocytes were obtained from hearts of neonatal Sprague–Dawlay rats. siRNAs were used to down-regulate RyR2 expression. Reduction of RyR2 expression was documented by RT-PCR, western blot, and immunofluorescence. Ca2+ signals were investigated by measuring the relative intracellular Ca2+ concentration, spontaneous Ca2+ oscillations, caffeine-induced Ca2+ release, and L-type Ca2+ currents. In normal cardiomyocytes, steady and periodic spontaneous Ca2+ oscillations were observed, and the baseline [Ca2+]i remained at the low level. Exposure to 3 mMaconitine increased the frequency and decreased the amplitude of Ca2+ oscillations; the baseline [Ca2+]i and the level of caffeine-induced Ca2+ release were increased but the L-type Ca2+ currents were inhibited after application of 3 mM aconitine for 5min. In RyR2 knockdown cardiomyocytes, the steady and periodic spontaneous Ca2+ oscillations almost disappeared, but were re-induced by aconitine without affecting the baseline [Ca2+]i level; the level of caffeine-induced Ca2+ release was increased but L-type Ca2+ currents were inhibited. Alterations of RyR2 are important consequences of aconitine-stimulation and activation of RyR2 appear to have a direct relationship with aconitine-induced arrhythmias. The present study demonstrates a potential method for preventing aconitine-induced arrhythmias by inhibiting Ca2+ leakage through the sarcoplasmic reticulum RyR2 channel.