Phosphofructokinase-2/fructose-2, 6-bisphosphatase 3 (PFKFB3) catalyzes the synthesis of F2,6BP, which is an allosteric activator of 6-phosphofructo-1-kinase (PFK-1): the rate-limiting enzyme of glycolysis. During tumorigenesis, PFKFB3 increases glycolysis, angiogenesis, and tumor progression. In this study, our aim was to investigate the significance of PFKFB3 and Ki67 in human lung adenocarcinomas and to target PFKFB3 as a therapeutic strategy. In this study, we determined the expression levels of PFKFB3 mRNA and proteins in cancerous and normal lung adenocarcinomas by quantitative reverse transcription PCR (qRT-PCR), Western blot analysis, and tissue microarray immunohistochemistry analysis, respectively. In human adenocarcinoma tissues, PFKFB3 and Ki67 protein levels were related to the clinical characteristics and overall survival. Both PFKFB3 mRNA and protein were significantly higher in lung adenocarcinoma cells (all P < 0.05). A high expression of PFKFB3 and Ki67 were associated with the degree of differentiation, TNM staging, lymph node metastasis, and survival. A high expression of PFKFB3 protein was an independent prognostic marker in lung adenocarcinoma. Subsequently, 1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one (PFK15) was used as a selective antagonist of PFKFB3. Glycolytic flux was determined by measuring glucose uptake, F2,6BP, and lactate production. Cell viability, cell cycle, cell apoptosis, cell migration, and invasion were analyzed by MTT, flow cytometry, Western blot analysis, wound healing assay, and transwell chamber assay. By targeting PFKFB3, it inhibited cell viability and glycolytic activity. It also caused apoptosis and induced cell cycle arrest. Furthermore, the migration and invasion of A549 cells was inhibited. We conclude that PFKFB3 bears an oncogene-like regulatory element in lung adenocarcinoma progression. In the treatment of lung adenocarcinoma, targeting PFKFB3 would be a promising therapeutic strategy.