Objective To explore the effects of the inhibition of Janus kinase 2/signal transducers and activator of transcription 3 (JAK2/STAT3) on the expression of hypoxia inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), and microRNA-145 (miRNA-145) in A549 lung cancer cell line. Methods The  expressions of HIF-1α, VEGF, and miRNA-145 protein and mRNA in A549 cells were detected by the Western blotting and RT-PCR after being treated by Jak2/STAT3 specific inhibitor AG490 of different concentrations (the AG490 group) or transfected by siRNA targeting to STAT (the siRNA-STAT group). Cells of the blank control group were not treated by AG490 or transfected. Cells of the negative control group were siRNA cells of sequences irrelevant to the transfection. Results Compared to the blank control group, the expression of miRNA-145 of A549 cells of the AG490 group up-regulated significantly (P<0.05) and the expressions of HIF-1α and VEGF protein and mRNA gradually decreased with the increase of the concentration of AG490. Compared to the blank control group and negative control group, the expressions of STAT3, phosphorylated STAT3, HIF-1α, and VEGF mRNA of A549 cells of the siRNA-STAT group down-regulated significantly, while the expression of miRNA145 increased significantly (P<0.05). Conclusion The inhibition of JAK2/STAT3 signaling pathway of A549 human lung adenocarcinoma cell can up-regulate the expression of miRNA-145 and down-regulate the expressions of HIF-1α and VEGF.