Regulation of Nuclear Pre-mRNA Domain Containing 1B (RPRD1B) has been of great interest in the field of oncology in recent years. The relationship between miRNAs and RPRD1B in gastric cancer (GC) has not been adequately reported. This study was designed to screen RPRD1B-targeted miRNAs and investigate its regulatory mechanism in GC cells. Quantitative RT-PCR and in situ hybridization were used to detect miRNA expression in GC tissues. Colony formation, EdU cell proliferation assay, and flow cytometry were used to analyze the cell cycle. Database-assisted gene expression analysis revealed that RPRD1B was targeted and regulated by miRNA-139-5p in GC. miRNA-139-5p expression was higher in GC tissue than in normal tissues and significantly correlated with tumor size, pathological stage, and disease-free survival of GC (p < 0.05). MiRNA-139-5p regulates GC cell proliferation and affects the transition from G1 to S phase. It binds explicitly to the 2013-2019 sites of the 3'UTR of RPRD1B and negatively regulates RPRD1B expression. We demonstrated that the ability of miR-139-5p to regulate GC cell proliferation depends on RPRD1B. This process is accompanied by changes in Cyclin D1 protein expression. We established a miR-139-5p/RPRD1B/tumor proliferation axis in GC, which may serve as novel biomarkers and drug targets for GC. (C) 2020 Elsevier Inc. All rights reserved.