G-rich oligonucleotides (GROs) belong to a novel class of phosphodiester oligonucleotides. They can form G-tetramer structure which contributes to cell cycle arrest and growth inhibitory effects by non-antisense pathway. This study was aimed to investigate the biological effects of GRO-26B on leukemia cell lines. Cell proliferation of different cell lines were detected by using MTT method and trypan blue incorporation assay. Alteration of cell cycle was analyzed by using flow cytometry. Apoptosis was detected by using Annexin V/PI kit. Western blot was used to detect the expression level of cyclins and CDKs. Morphological features of GRO-26B-treated cells was observed by light microscopy and transmission electron microscopy (TEM). The results showed that GRO-26B could inhibit the proliferation of AML cell lines, such as U937 and NB4 cells in a dose-dependent manner. GRO-26B induced the cell cycle to be arrested at S phase in time-dependent manner, which was associated with the alteration of cyclin A, cyclin B, CDC2 and CDK2. The morphology of cells treated by GRO-26B also showed a distinct change as compared to the untreated cells. It is concluded that GRO-26B can inhibit AML cell proliferation, which is partially associated to cell cycle arrest at S phase. The S phase arrest is related to cyclins/CDKs. The regulation mechanism of cell cycle and proliferation is complicated. All of the above-mentioned phenomena need to be studied in the future.