Background: Angiogenesis is the growth of new blood vessels from preexisting vascular structures. Clinical trials using vascular endothelial growth factor 165a (VEGF165a) for angiogenesis in peripheral arterial disease (PAD) yielded little success. Glycolysis accounts for 85% of ATP production in endothelial cells (ECs) and is further activated during VEGF165a induced angiogenesis. Our previous research demonstrated interleukin 21 (IL21) promoting angiogenesis in PAD models independently of the VEGF165a pathway. Hypothesis: IL21 utilizes different metabolic pathways than glycolysis in ECs under ischemic conditions. Goal: Explore the metabolic pathway(s) activated in ECs in response to IL21 and its functional consequence on angiogenesis in preclinical models of PAD. Methods: Human umbilical vein endothelial cells (HUVECs) were cultured under hypoxia and serum starvation media, treated with PBS (control), VEGF165a (50 ng/mL), or IL21 (50 ng/mL). Methods included qPCR, Western blot (WB) of the genes of the rate controlling enzymes of the main metabolic pathways , angiogenesis assay tube formation on Matrigel and assessing endothelial barrier function using trans endothelial resistance (TER). Results: In HUVECs under HSS, using qPCR, IL21 vs. PBS upregulates glutamine fructose-6-phosphate amido-transferase isoform 1 (GFAT1); the rate limiting enzyme for hexosamine biosynthesis pathway (HBP) (Relative expression 1.28±0.08 SD vs. 1.00±0.04 SD, n=3/group, p=0.03) while VEGF165a did not (Relative expression 1.04±0.03 SD vs. 1.00±0.04 SD, n=3, p=0.23). WB showed similar finding where IL21 vs. PBS increased GFAT1 expression under the same conditions (Relative expression 1.74±0.39 SD vs. 1.00±0.16 SD, n=3/group, p=0.04) while VEGF165a did not. Knocking down GFAT1 reduced IL21 mediated hypoxia dependent angiogenesis (number of tubes, 1.00±0.37 SD vs. 0.37±0.10 SD, n=4-5/group, p<0.0001) but not VEGF165a. The activation of HBP by IL21, was associated with a more stable barrier function compared to VEGF165a (TER relative to control; 0.87±0.01 vs. 0.79±0.01, n=5/group, p<0.0001). Conclusion: These results unmask an unexpected role of HBP in angiogenesis regulation, emphasizing the significance of EC metabolism under PAD conditions.