Objective: To investigate the effect of honokiol on the growth of gallbladder cancer cells in vitro and its mechanism. Methods: Using CCK-8 assay, the inhibitory effect of honokiol on gallbladder cancer SGC-996 cells was observed and the 50% inhibitory concentration (IC50) values were calculated. After exposure of SGC-996 cells to different concentrations of honokiol for 48 h, the colony formation, apoptosis and cell cycle as well as the expressions of apoptosis and cell cycle related proteins were examined by plate colony formation assay, cytometry and Western blot analysis, respectively. Results: Honokiol significantly inhibited the growth of SGC-996 cells, and its IC50 value was 34.66, 23.20 and 18.87 μmol/L after 24, 48 and 72 h, respectively. After exposure of SGC-996 cells to honokiol, the number of colony forming units was decreased, apoptosis rate and percentage of G0/G1 cells were increased, and the expressions of pro-apoptotic proteins (Bax, cleaved-caspase-9, cleaved-caspase-3 and cleaved-PARP) were increased, while expressions of anti-apoptotic proteins (Bcl-2 and Bcl-2/Bax ratio) as well as cell cycle-related proteins (Cyclin D1, Cdk4 and Cdk6) were decreased. All of these effects showed significant concentration-dependent trend (all P<0.05). Conclusion: Honokiol has strong inhibitory effect on gallbladder cancer cells in vitro, and its mechanism may possibly be associated with inducing cell apoptosis via intrinsic apoptosis pathway and suppressing cell proliferation by regulating the expression of cell cycle-related proteins.