In chronic lymphocytic leukemia, the hypoxia-inducible factor 1 (HIF-1) regulates the response of tumour cells to hypoxia and their protective interactions with the leukemic microenvironment. In this study we demonstrate that chronic lymphocytic leukemia cells from TP53-disrupted (TP53dis) patients have constitutively higher expression levels of the α-subunit of HIF-1 (HIF-1 α) and increased HIF-1 transcriptional activity, compared to the wild type counterpart. In the TP53dis subset, HIF-1 α upregulation is due to reduced expression of the HIF-1 α ubiquitin ligase von Hippel-Lindau protein (pVHL). Hypoxia and stromal cells further enhance HIF-1α accumulation, independently from the TP53 status. Hypoxia acts through the downmodulation of pVHL and the activation of the PI3K/AKT and RAS/ERK1-2 pathways, whereas stromal cells induce an increased activity of the RAS/ERK1-2, RHOA/RHOA kinase and PI3K/AKT pathways, without affecting pVHL expression. Interestingly, we observed that higher levels of HIF-1A mRNA correlate with a lower susceptibility of leukemic cells to spontaneous apoptosis, and associate with the fludarabine resistance that mainly characterizes TP53dis tumour cells. The HIF-1α inhibitor BAY87-2243 exerts cytotoxic effects toward leukemic cells, regardless of the TP53 status, and has anti-tumour activity in Eμ-TCL1 mice. BAY87-2243 also overcomes the constitutive fludarabine resistance of TP53dis leukemic cells and elicits a strongly synergistic cytotoxic effect in combination with ibrutinib, thus providing preclinical evidences for its further investigation as a potential new drug in chronic lymphocytic leukemia.