PFKFB (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase) catalyses the synthesis and degradation of Fru-2,6-P2 (fructose-2,6-bisphosphate), a key modulator of glycolysis and gluconeogenesis. The PFKFB3 gene is extensively involved in cell proliferation owing to its key role in carbohydrate metabolism. In the present study we analyse its mechanism of regulation by progestins in breast cancer cells. We report that exposure of T47D cells to synthetic progestins (ORG2058 or norgestrel) leads to a rapid increase in Fru-2,6-P2 concentration. Our Western blot results are compatible with a short-term activation due to PFKFB3 isoenzyme phosphorylation and a long-term sustained action due to increased PFKFB3 protein levels. Transient transfection of T47D cells with deleted gene promoter constructs allowed us to identify a PRE (progesterone-response element) to which PR (progesterone receptor) binds and thus transactivates PFKFB3 gene transcription. PR expression in the PR-negative cell line MDA-MB-231 induces endogenous PFKFB3 expression in response to norgestrel. Direct binding of PR to the PRE box (−3490 nt) was confirmed by ChIP (chromatin immunoprecipiation) experiments. A dual mechanism affecting PFKFB3 protein and gene regulation operates in order to assure glycolysis in breast cancer cells. An immediate early response through the ERK (extracellular-signal-regulated kinase)/RSK (ribosomal S6 kinase) pathway leading to phosphorylation of PFKFB3 on Ser461 is followed by activation of mRNA transcription via cis-acting sequences on the PFKFB3 promoter.