Chemotherapy has been widely used to treat cancer, however, the appearance of multiple drug resistance (MDR) in cancer patients is regarded as a major clinical obstacle to successful chemotherapy. MicroRNAs (miRNAs) are evolutionary conserved small RNAs that regulate gene expression at the post-transcriptional level and have been shown to regulate cell differentiation, development, proliferation and apoptosis. Nevertheless, the involvement of miRNAs and their roles in the development of MDR in liver cancer are not fully understood. Our study found that the expression of miR-27a was down-regulated in the multidrug-resistant hepatocellular carcinoma cell line BEL-7402/5-fluorouracil (BEL/5-FU) compared with its parental BEL-7402 cell line, while the MDR1/P-glycoprotein expression was elevated. Overexpression of miR-27a by transfecting with miR-27a mimics in the BEL/5-FU cells could reduce the MDR1/P-glycoprotein and β-catenin expressions, enhance the sensitivity of these cells to 5-fluorouracil and 5-fluorouracil-induced apoptosis. Moreover, up-regulation of miR-27a did not decrease the FZD7 mRNA level, but significantly reduce its protein expression in BEL/5-FU cells. It was also confirmed that reduction of FZD7 by RNA interference induced inhibitory effects on the expression of MDR1/P-glycoprotein and β-catenin, similar to miR-27a. Taken together, our findings suggest that miR-27a could function as a novel regulator to reverse MDR in hepatocellular carcinoma cells by inhibiting the FZD7/β-catenin pathway.