Most cancer cells utilize glucose at a high rate to produce energyand precursors for the biosynthesis of macromolecules such as lipids, proteins, and nucleic acids. This phenomenon is called the Warburg effect or aerobic glycolysis- this distinct characteristic is an attractive target for developing anticancer drugs. Here, we found that Phosphofructokinase-1 (PFK-1) is a substrate of the Protein Phosphatase 4 catalytic subunit (PP4C)/PP4 regulatory subunit 1 (PP4R1) complex by using immunoprecipitation and in vitro assay. While manipulation of PP4C/PP4R1 does not have a critical impact on PFK-1 expression, the absence of the PP4C/PP4R1 complex increases PFK-1 activity. Although PP4C depletion or overexpression does not cause a dramatic change in the overall glycolytic rate, PP4R1 depletion induces a considerable increase in both basal and compensatory glycolytic rates, as well as the oxygen consumption rate, indicating oxidative phosphorylation. Collectively, the PP4C/PP4R1 complex regulates PFK-1 activity by reversing its phosphorylation and is a promising candidate for treating glycolytic disorders and cancers. Targeting PP4R1 could be a more efficient and safer strategy to avoid pleiotropic effects than targeting PP4C directly. [BMB Reports 2023; 56(11): 618-623].