AIM: To investigate the expression of toll-like receptor (TLR) 4, nuclear factor-κB (NF-κB) p65 and hypoxiainducible transcription factor 1α (HIF-1α) in pancreatic ductal adenocarcinoma and their clinical significance. METHODS: The mRNA of TLR4 and HIF-1α were investigated by real-time polymerase chain reaction in 30 cases of pancreatic ductal adenocarcinoma and its adjacent tissues, and expression of TLR4, NF-κB p65 and HIF-1α protein were detected by immunohistochemistry in 65 cases of pancreatic ductal adenocarcinoma tissues and 38 cases of corresponding adjacent tissues. The relationship between TLR4 or HIF-1α and pathologic features, as well as the association between TLR4 and HIF-1α, were also analyzed. Kaplan-Meier method was used to assess the impact of expression of TLR4 and HIF-1α on survival of patients with pancreatic cancer. RESULTS: The relative quantification of TLR4 and HIF-1α mRNA in tumor tissues was 0.81 ± 0.10 and 0.87 ± 0.11, respectively, significantly higher than that in adjacent tissues (0.81 ± 0.10 vs 0.70 ± 0.16, P = 0.002; 0.87 ± 0.11 vs 0.68 ± 0.13, P = 0.000). The protein expression of TLR4, NF-κB p65 and HIF-1α in tumor tissues was 69.20%, 66.15% and 70.80%, respectively, being significantly higher than that in adjacent normal tissues (69.20% vs 39.50%, P = 0.003; 66.15% vs 31.58%, P = 0.001; 70.80% vs 36.80%, P = 0.001). There was no significant correlation between TLR4 or HIF-1α expression and the age, gender, tumor location, the degree of tumor differentiation in the patients ( P > 0.05). However, there was significant correlation between the expression of TLR4 or HIF-1α and tumor size, lymph node metastasis, venous invasion and clinical staging ( P < 0.05). The expression of TLR4 and HIF-1α had a significant impact on survival of patients with pancreatic adenocarcinoma. CONCLUSION: TLR4, NF-κB p65 and HIF-1α are overexpressed in pancreatic adenocarcinoma, TLR4 may be partly involved in up-regulating HIF-1α, and both synergestically promote development of pancreatic adenocarcinoma.