Objective To observe the expressions of farnesol X receptor (FXR)/nuclear factor receptor 1H4 (NR1H4) mRNA and protein in pancreatic ductal adenocarcinoma tissues, and to analyze their correlations with clinicopathological features and prognosis. Methods A total of 176 pancreatic ductal adenocarcinoma specimens were collected from Changhai Hospital of Naval Medical University (Second Military Medical University) from 2013 to 2016. The clinicopathological data of the patients were collected. All the specimens were made into tissue microarray. The expression of NR1H4 mRNA was detected by RNAscope in situ hybridization and NR1H4 probe, and FXR/NR1H4 protein was detected by polymer two-step immunohistochemistry. Chi-square test was used to analyze the relationship between the expression levels of FXR/NR1H4 mRNA and protein and clinicopathological features. Kaplan-Meier survival analysis and Cox regression model were used to study the relationship between the expression levels of FXR/NR1H4 mRNA and protein and patient prognosis. Results The positive rate of NR1H4 mRNA detected by RNAscope in situ hybridization was 68.75% (121/176), and that of FXR/NR1H4 protein detected by immunohistochemistry was 77.27% (136/176). FXR/NR1H4 mRNA and protein expression levels were correlated with clinical stage (χ²=5.391, P=0.020; χ²=4.108, P=0.042) and differentiation (χ²=6.560, P=0.010; χ²=4.969, P=0.026); and the protein expression was also related to tumor size (χ²=4.957, P=0.026). No correlations were found between FXR/NR1H4 expression and the patient gender, age, nerve invasion, tumor location or lymph node metastasis (all P>0.05). Kaplan-Meier survival analysis showed that the progression-free survival and overall survival of patients with high expression levels of FXR/NR1H4 mRNA and protein were shorter than those with low expression levels (P<0.05). Cox regression analysis showed that high expression of FXR/NR1H4 protein was an independent risk factor of progressionfree survival (RR=1.701, 95% CI 1.235-2.432, P<0.05) and overall survival of patients (RR=2.356, 95% CI 1.983-2.832, P<0.05). Conclusion RNAscope in situ hybridization and immunohistochemistry can confirm each other to ensure the reliability of the detection results. The expression of FXR/NR1H4 is related to the clinical stage and differentiation of pancreatic ductal adenocarcinoma, and its high expression is an independent risk factor of prognosis.