Objective To evaluate the role of glycogen synthase kinase 3 beta (GSK-3β) in lipid emulsion-induced inhibition of bupivacaine-induced apoptosis in cardiomyocytes of rats using RNA interference (RNAi) adenovirus infection method. Methods H9C2 cells were transferred into 96-well cell plates at a density of 1×105 cells/ml after culture and then divided into 8 groups (n=10 each) using a random number table: control group (group C), bupivacaine group (group B), lipid emulsion group (group LE), bupivacaine plus lipid emulsion group (group B+ LE), control plus GSK-3βRNAi adenovirus (GSK-3βi) group (group C+ GSK-3βi), bupivacaine plus GSK-3βi group (group B+ GSK-3βi), lipid emulsion plus GSK-3βi group (group LE+ GSK-3βi) and bupivacaine plus lipid emulsion plus GSK-3βi group (group B+ LE+ GSK-3βi). In B, LE and B+ LE groups, the cells were incubated with culture medium containing 1 mmol/L bupivacaine, 1% lipid emulsion and 1 mmol/L bupivacaine plus 1% lipid emulsion, respectively.In C+ GSK-3βi, B+ GSK-3βi, LE+ GSK-3βi and B+ LE+ GSK-3βi groups, the cells were incubated with the drugs mentioned above on 2nd day after being infected by adenovirus.At 24 h after incubation with drugs, the expression of Bax and Bcl-2 was determined by Western blot, and the apoptosis rate was calculated using DAPI staining. Results Compared with group C, the expression of Bax was significantly up-regulated, the expression of Bcl-2 was down-regulated, and the apoptosis rate was increased in group B (P<0.05). Compared with group B, the expression of Bax was significantly down-regulated, the expression of Bcl-2 was up-regulated, and the apoptosis rate was decreased in group B+ LE (P<0.05). Compared with group B+ LE, the expression of Bax was significantly up-regulated, the expression of Bcl-2 was down-regulated and the apoptosis rate was increased in group B+ LE+ GSK-3βi (P<0.05). Conclusion The mechanism by which lipid emulsion inhibits bupivacaine-induced apoptosis in cardiomyocytes of rats is associated with GSK-3β. Key words: Fat emulsions, intravenous; Bupivacaine; Apoptosis; Myocytes, cardiac