Pancreatic cancer (PC) can be considered a representative cancer type of the human body. As demonstrated by some studies, microRNA (miR)-499 is dysregulated in various cancer types including PC, for which chemotherapy involving 5-fluorouracil (5-FU) has long been considered the first-line therapy. However, there are complex and comprehensive mechanisms related to 5-FU, which have not been fully elucidated. This study thus aimed to examine the molecular mechanisms of 5-FU resistance through miR-499a-5p in PC. The expression of miR-499a-5p in PC was measured using quantitative polymerase chain reaction (PCR). MiR-499a-5p was examined in-vivo for its effects on the malignant phenotypes of PC cells. The results of the present study demonstrated miR-499a-5p to be upregulated in PC and 5-FU resistant PC tissues. According to in vitro assays in PC cells (PANC1/FR), miR-499a-5p was found to affect adenosine triphosphate (ATP) binding cassette subfamily B member 1 (P-gp), ATP binding cassette subfamily C member 1 (MRP1), and ATP binding cassette subfamily G member 2 (BCRP), thereby facilitating 5-FU resistance in PC cells. Functions assays indicated that suppressed miR-499a-5p expression inhibited the proliferation and migration of cells but facilitated apoptosis in the PC cell line; by contrast, miR-499a-5p overexpression triggered the inverse phenotypic changes of cells. Concerning the mechanisms involved, miR-499a-5p increased PI3K/Akt signaling by targeting phosphatase and tensin homolog (PTEN). Taken together, these findings demonstrate that miR-499a-5p can be potentially applied to PC therapy. 2021 Annals of Translational Medicine. All rights reserved.