The aim of study was to discuss the correlation and regulatory mechanism of HIF-1 and miR-191 expression in pancreatic tumor. The association between the miR-191 and the clinicopathologic characteristics and the prognosis of pancreatic cancer was further explored. After hypoxic cultured for 6 and 12 h, qRT-PCR and Western blot were practiced to analyze the miR-191 and HIF-1 expression of MIA PaCa-2 and Aspac1 cells. We regulated the HIF-1 expression via plasmid and siRNA transfection to observe the alteration of HIF-1 and miR-191 expression. ChIP sequencing identified the binding sites of HIF-1 and miR-191. Dual luciferase assays were practiced to verify the binding sites. Immunohistochemical staining was practiced to analyze the expression of HIF-1, while qRT-PCR were done for investigating miR-191 in tumor tissues. Then, the association between the expression of them and the clinicopathologic characteristics and prognosis of pancreatic cancer were analyzed. After hypoxic cultured 12 h, the expression of HIF-1 protein, HIF-1mRNA and miR-191 of MIA PaCa-2 and AsPC-1 cells increased significantly (P < 0.05). After HIF-1 overexpressing plasmid transfected to the MIA PaCa-2 and AsPC-1 cells for 48 h, the expression of HIF-1 protein, HIF-1mRNA, and miR-191 upregulated significantly (P < 0.05). While after transfected the MIA PaCa-2 cells by HIF-1 siRNA, the significant decreasing of HIF-1 protein, HIF-1mRNA, and miR-191 expression were observed (P < 0.05). ChIP sequencing showed the protein synthesis of HIF-1 increased in hypoxia situation. Only the HRE5 (-1,169 bp, ChIP4) were significantly brighter in hypoxia in comparing with normoxic cells. In dual luciferase assays, after pGL3-miR-191 and HIF-1 overexpressing plasmid co-transfect the MIAPaCa-2 cells for 48 h, its relative expression of bioluminescence was higher than those co-transfected by mutant miR-191 vectors and HIF-1 overexpressing plasmid or by pGL3-miR-191 and HIF-1 empty plasmid. The expression of miR-191 closely associated with the tumor size, pTNM stage, lymph node metastasis, and perineural invasion (P < 0.05). Patients with higher expression of miR-191 were a risk factor for prognosis of pancreatic cancers. Expression of HIF-1 in pancreatic cancer cells increased under the condition of chronic hypoxia, which may bind to HRE2 in 5'flanking region of miR-191 and initiate transcription of miR-191. Expression of miR-191 was significantly higher in pancreatic tumor tissues. The expression of miR-191 closely associated with the tumor size, pTNM stage, lymph node metastasis and perineural invasion and poor prognosis of pancreatic cancer.