Abstract: Background and purpose: Hepatocellular carcinoma is a common malignant tumor in clinic. Because of its tolerance to chemotherapeutic drugs, it often leads to poor prognosis. MicroRNA (miRNA, miR) is a family of small long non-coding RNA molecules, and dysregulated miRNA is associated with various tumor biological functions. MicroRNA-27a-3p has been extensively studied as a metabolism-related miRNA, but there are few reports on the relationship between miRNA and chemosensitivity. This study was focused on the effect of miR-27a-3p on chemoresistance in hepatocellular carcinoma to provide novel theoretical basis for liver cancer chemotherapy. Methods: The Cancer Genome Atlas (TCGA) database was used to analyze the expression of miR-27a-3p in hepatocellular carcinoma and adjacent tissues. Using liposome transfection method, miR-27a-3p overexpression plasmid and negative control plasmid (miR-control) were transfected into hepatocellular carcinoma cell line HepG2. miR-inhibitor-27a-3p knockdown plasmid and negative control plasmid (miR-inhibitor-control) were transfected into PLC cells. Then the cells were treated with different concentrations of cisplatin (DDP) for 48 h. MTT assay was used to detect the proliferation, and flow cytometry was used to detect the apoptosis and cell cycle. Western blot technique was used to detect the protein expression in PI3k/AKT signaling pathway. Results: The expression of miR-27a-3p in hepatocellular carcinoma tissue was significantly lower than that of the adjacent tissues (P<0.05). In miR-27a overexpressed HepG2 cells, MTT results showed that the half inhibitory concentration (IC ⁵⁰ ) of DDP in miR-27a+DDP group ［(1.02±0.03) μg/mL］ was significantly decreased compared with miR-control+DDP group [ (1.27±0.08) μg/mL] and the blank+DDP group [ (1.73±0.08) μg/mL] (P<0.05). The apoptosis results showed that miR-27a+DDP group ［(31.03±0.20)%］ had significantly increased apoptotic rate compared with miR-control+DDP group［(18.51±2.96)%］ and the blank+DDP group［(8.73±1.58)%］ (P<0.05). Western blot results showed that the PI3K expression level (0.28±0.01) in the miR- 27a+DDP group was significantly decreased compared with DDP group (0.43±0.01), miR-27a group (0.57±0.11) and miR-control group (0.72±0.01) (P<0.05). The expression level of p-AKT in miR-27a+DDP group (0.29±0.01) was significantly lower compared with DDP group (0.46±0.05), miR-27a group (0.67±0.01) and miR-control group (0.10±0.01) (P<0.05). The expression level of C-caspase in miR-27a+DDP group (0.69±0.01) was significantly higher compared with DDP group (0.51±0.01), miR-27a group (0.35±0.01) and miR-Con group (0.18±0.01) (P<0.05). miR-27a-3p was knocked down in the PLC cells which were cultured in DDP. The results of MTT, cell apoptosis and Western blot showed that the results were contrary to the results of HepG2 overexpressed cells, and the difference was statistically significant (P<0.05). Conclusion: miR-27a-3p may regulate the chemotherapy sensitivity to DDP in hepatocellular carcinoma cell lines by regulating the PI3K/AKT signaling pathway, which is expected to be a potential therapeutic target for enhancing the chemotherapy sensitivity to DDP in hepatocellular carcinoma.