A natural α-1,6-glucan named BBWPW was identified from black beans. Cell viability assay showed that BBWPW inhibited the proliferation of different cancer cells, especially HeLa cells. Flow cytometry analysis indicated that BBWPW suppressed the HeLa cell cycle in the G2/M phase. Consistently, RT-PCR experiments displayed that BBWPW significantly impacts the expression of four marker genes related to the G2/M phase, including p21, CDK1, Cyclin B1, and Survivin. To explore the molecular mechanism of BBWPW to induce cell cycle arrest, a transcriptome-based target inference approach was utilized to predict the potential upstream pathways of BBWPW and it was found that the PI3K-Akt and MAPK signal pathways had the potential to mediate the effects of BBWPW on the cell cycle. Further experimental tests confirmed that BBWPW increased the expression of BAD and AKT and decreased the expression of mTOR and MKK3. These results suggested that BBWPW could regulate the PI3K-Akt and MAPK pathways to induce cell cycle arrest and ultimately inhibit the proliferation of HeLa cells, providing the potential of the black bean glucan to be a natural anticancer drug.