Treatment of patients with locally advanced rectal cancer (RC) is based on neoadjuvant chemoradiotherapy followed by surgery. In order to reduce the development of therapy resistance, it is necessary to further improve previous treatment approaches. Recent in vivo experimental studies suggested that the reduction of tumor hypoxia by tumor vessel normalization (TVN), through the inhibition of the glycolytic activator PFKFB3 could significantly improve tumor response to therapy. We have evaluated in vitro and in vivo the effects of the PFKFB3 inhibitor 2E-3-(3-Pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) on cell survival, clonogenicity, migration, invasion and metabolism using colorectal cancer cells, patient-derived tumor organoids (PDO) and xenografts (PDX). 3PO treatment of colorectal cancer cells increased radiation-induced cell death and reduced cancer cell invasion. Moreover, Gene Set Enrichment Analysis shows that 3PO is able to alter the metabolic status of PDOs towards oxidative phosphorylation. Additionally, in vivo neoadjuvant treatment with 3PO induced TVN, alleviated tumor hypoxia and increased tumor necrosis. Our results support PFKFB3 inhibition as a possible future neoadjuvant addition for rectal cancer patients.