The expression of hTERT in tumor cells contributes to oncogenic transformation by promoting immortalization. For this reason, hTERT is one of the major targets for cancer therapy, and an efficient method to downregulate hTERT expression is required for treatment of hTERT-positive cancer. In this report, we demonstrated that inhibition of AMP-activated protein kinase (AMPK) downregulates the expression of hTERT. We screened cell signaling pathways in AMPK α1 knockout cells and found that AMPKα1 is required for activity of the hTERT promoter. AMPKα1 knockout cells showed decreased expression of hTERT mRNA and protein. We also demonstrated that compound C, a reversible AMPK inhibitor, suppressed the expression of hTERT. However, AMPK activators, including AICAR and metformin, did not increase the level of hTERT protein. Finally, we showed that tumor cells stably expressing hTERT are resistant to compound C treatment. These results indicate that AMPK activity is required for tumor progression.