CD133 is a cellular surface protein, which has been reported to be a cancer stem cell marker, and thus is considered a potential target for cancer treatment. Metformin is also known to reduce the risk of cancer development and cancer stem cells (CSCs), including the expression of CD133. However, the mechanism underlying the reduction of the expression of CD133 by metformin is not yet understood. This study shows that metformin suppressed CD133 expression mainly by affecting the CD133 P1 promoter via AMPK signaling. AMPK inhibition rescued the reduction of CD133 by metformin. Further experiments demonstrated that CEBPβ was upregulated by metformin and that two isoforms of CEBPβ reciprocally regulated the expression of CD133. Specifically, the liver-enriched activator protein (LAP) isoform increased the expression of CD133 by directly binding to the P1 promoter region, whereas the liver-enriched inhibitory protein (LIP) isoform suppressed the expression of CD133. Consistent with these findings, a 3D culture assay and drug sensitivity assay demonstrated that LAP-overexpressing cells formed large spheroids and were more resistant to 5-FU treatment, whereas LIP-overexpressing cells were more sensitive to 5-FU and showed combined effects with metformin. Our results indicated that metformin-AMPK-CEBPb signaling plays a crucial role in regulating the gene expression of CD133. Additionally, regulating the ratio of LAP/LIP may be a novel strategy for targeting CSCs for the treatment of cancer.