The drug-resistance of pancreatic cancer cells results in poor therapeutic effect. To predict the therapeutic effect of the chemotherapy drugs to specific patients and to reverse the resistance of pancreatic cancer cells are critical for chemotherapy of pancreatic cancer. MicroRNAs (miRNAs) have been reported to play important roles in the genesis of drug-resistance of various cancer types. There are also many advantages of miRNAs in diagnosis and therapy of disease. Although several miRNAs regulating 5-Fluorouracil (5-FU) resistance in human pancreatic cancer have been reported, the detailed molecular mechanism remains to be determined. In this study, we found that miR-320a was significantly up-regulated in 5-FU resistant pancreatic cancer cells. Over-expression of miR-320a strongly contributed to pathogenesis of pancreatic cancer, which was represented by the increased proliferation, invasion, metastasis, drug-resistance characteristics and the epithelial-to-mesenchymal transition. Furthermore, we demonstrated that miR-320a was able to bind to 3'UTR of PDCD4 mRNA, and mediated its down-regulation in 5-FU resistance of human pancreatic cancer cells. Whereas restoration of PDCD4 expression could partially attenuate the function of miR-320a in pancreatic cancer. Taken together, our study demonstrated that miR-320a played important role in regulating 5-FU resistance by targeting PDCD4 and might be developed as new therapeutic target for pancreatic cancer.