Objectives：To reveal the relationship between microRNA-10b and gemcitabine (GEM) resistance of pancreatic cancer and its possible mechanism. Methods: The expression of microRNA-10b in PANC-1 cells (which are relatively resistant to GEM) and CFPAC-1 cells (which are relatively sensitive to GEM) were detected by RT-qPCR. PANC-1 and CFPAC-1 cells were treated with different doses of GEM，and then the cells were harvested. The expression of microRNA-10b was detected by RT-qPCR. CCK-8 assay and flow cytometry were performed to evaluate the drug sensitivity of CFPAC-1 cells，which were transfected with microRNA-10b mimics to GEM. The protein expressions of PI3K，p-Akt，Bcl-2 and Survivin in CFPAC-1 cells transfected with microRNA-10b mimics or negative control were demonstrated using Western blotting assay. The gene expressions of PTEN in CFPAC-1 cells transfected with microRNA-10b mimics or negative control were detected by RT-qPCR. Results: PANC-1 cells showed significantly increased expression of microRNA-10b as compared to CFPAC-1 cells. The expression of microRNA-10b both in PANC-1 and CFPAC-1 cells increased with the rising of GEM concentration. The sensitivity of CFPAC-1 cells to GEM decreased when microRNA-10b was overexpressed and the expressions of PI3K，p-Akt，Bcl-2，and Survivin were much higher than their control groups. The expression of PTEN mRNA decreased as microRNA-10b increased. Conclusions: MicroRNA-10b might negatively regulate the gene expression of PTEN，which increases the protein expressions of PI3K，p-Akt，Bcl-2 and Survivin，thus reduces the sensitivity of CFPAC-1 cells to GEM，and as a result，pancreatic cancer cells become resistant to GEM.