Metformin has been known to suppress cancer stem cells (CSCs) in some cancers. However, the differential effects of metformin on CSCs and their mechanisms have not been reported. Herein, metformin induced pAMPK activation and pS6 suppression in metformin-sensitive (HT29) cells, but not in metformin-resistant (SW620) cells. The oxygen consumption rate was higher in HT29 cells than in SW620 cells and showed a prominent decrease after metformin treatment in HT29 cells. In glutamine-depleted medium, but not in low-glucose medium, SW620 cells became sensitive to the CSC-suppressing effect of metformin. A combination of metformin and glutaminase C inhibitor (compound 968) suppressed CSCs in SW620 cells and enhanced that effect in HT29 cells. SW620 cells showed higher expression of glutaminase 1 and glutamine transporter (ASCT2) than HT29 cells, especially ASCT2 in CSCs. Knockdown of glutaminase 1, ASCT2, and c-Myc induced significant CSC-suppression and enhanced CSC-suppressing effect of metformin and compound 968. In xenografts and human cancer organoids, combined treatment with metformin and compound 968 showed the same results as those shown in vitro. In conclusion, the effect of metformin on CSCs varies depending on the AMPK-mTOR and glutamine metabolism. The inhibition of glutamine pathway could enhance the CSC-suppressing effect of metformin, overcoming metformin resistance.