Polydatin (PD), a natural precursor of resveratrol, has a variety of biological activities, including anti-tumor effects. However, the underlying molecular mechanisms of the anti-cancer activity of PD has not been fully elucidated. The present study demonstrated that PD significantly inhibited the proliferation of the MOLT-4 leukemia cell line in a dose- and time-dependent manner by using Cell Counting Kit-8 assay. PD also dose-dependently increased the apoptotic rate and caused cell cycle arrest in S phase in MOLT-4 cells, as revealed by flow cytometry. In addition, PD dose-dependently decreased the mitochondrial membrane potential and led to the generation of reactive oxygen species in MOLT-4 cells. Western blot analysis revealed that the expression of anti-apoptotic protein B-cell lymphoma 2 ( Bcl-2) was decreased, whereas that of pro-apoptotic protein Bcl-2-associated X was increased by PD. Furthermore, the expression of two cell cycle regulatory proteins, cyclin D1 and cyclin B1, was suppressed by PD. Of note, the pro-apoptotic and cell cycle-inhibitory effects of PD were potentiated by Janus kinase (JAK2) inhibition. In conclusion, the results of the present study strongly suggested that PD is a promising therapeutic compound for the treatment of leukemia, particularly in combination with JAK inhibitors.