The effect of timosaponin AIII (TSA-III) on human leukaemia HL-60 cells and the underlying mechanism of action were explored in this study. HL-60 cells were treated with different concentrations of TSA-III, and then the survival rate and apoptosis rate of the cells, expression of apoptosis-related genes, activation of caspase-3, cell distribution in the cell cycle phases, and expression level of the related signal pathway proteins were detected. The results showed that TSA-III could dose- and time-dependently inhibit the proliferation of HL-60 cells. TSA-III could also increase the apoptosis rate of HL-60 cells, the level of Bax and Bak mRNA and the activation rate of caspase-3, and decrease the level of Bcl-2 mRNA. At the same time, the proportion of HL-60 cells treated with TSA-III in G0/G1 phase increased, whereas the proportion of the cells in S phase decreased. In addition, TSA-III could elevate the expression of PTEN protein, and reduce the expression of p-AKT, beta-catenin, Cyclin D1 and C-myc proteins. These results indicate that TSA-III should have a cytotoxic effect on HL-60 cells, mainly showing its inhibition on the proliferation, inducing the apoptosis and cell cycle arrest, and the mechanism may be related to its inhibition of PI3K/AKT and Wnt/beta-catenin pathways.