Hypoxic microenvironment deregulates metabolic homeostasis in cancer cells albeit the underlying mechanisms involved in this process remain hitherto enigmatic. 14-3-3 zeta/Yes-associated protein (YAP) axis plays a principal role in malignant transformation and tumor development. Here, we report that hypoxia disassembles 14-3-3 zeta from YAP and thereby promotes YAP nuclear localization mediated by ERK2, which directly binds to the D-site of mitogen-activated protein kinase (MAPK) docking domain in 14-3-3 zeta Leu98/100 and phosphorylates 14-3-3 zeta at Ser37. When localizing in nucleus, YAP recruits at pyruvate kinase M2 (PKM2) gene promoter with hypoxia-inducible factor 1 alpha (HIF-1 alpha), for which PKM2 transcription is required. 14-3-3 zeta Ser37 phosphorylation is instrumental for the hypoxia-induced glucose uptake, lactate production, and clonogenicity of pancreatic ductal adenocarcinoma (PDAC) cells, as well as tumorigenesis in mice. The 14-3-3 zeta Ser37 phosphorylation positively correlates with p-ERK1/2 activity and HIF-1 alpha expression in clinical samples from patients with PDAC and predicts unfavorable prognosis. Our findings underscore an appreciable linkage between YAP transcriptional activation and hypoxic glycolysis governed by ERK2-dependent 14-3-3 zeta Ser37 phosphorylation for malignant progression of PDAC.