Objective · To observe real-time changes of calcium concentration ([Ca2+]i) exposure to bilirubin in synaptosomes isolated from brainstem nucleus of rats.  Methods · Forty P7-14 SD rats were randomly assigned to three groups: control group, bilirubin group (with levels of 0.1, 1 and 10 μmol/L) and bulirubin plus glycoursodeoxycholic acid (GUDCA) group. The synaptosomes were purified from brainstem nucleus by sucrose density gradient centrifugation. After loading OG-BAPTA in synaptosomes, two dimensional image of intracellular calcium and analysis of fluorescence intensity were achieved by Confocal laser scanning microscopy.  Results · Synaptosomes with well biological activity were obtained from brainstem of the SD rats. In the control group, a progressive increase in fluorescent intensity of [Ca2+]i was detected. In the bilirubin group, acuter increases in fluorescent intensity were observed in all levels of bilirubin, with a manner of both concentration and time-dependent (P<0.05). Fluorescent intensity of [Ca2+]i was reduced  in the present of GUDCA, which was not significant compared with the control group (P=0.656). However, GUDCA could abate the increase of fluorescent intensity of [Ca2+]i induced by bilirubin exposure, of which showing significant decrease in 10 μmol/L bilirubin exposure (P=0.000).  Conclusion · Bilirubin could induce calcium overload in synaptosomes. GUDCA could abate bilirubin-induced calcium overload in synaptosomes, possibly explaining its protection effect of neurons from bilirubin neurotoxicity.