Objective To investigate the effect of microRNA (miRNA)-544-3p on hepatitis B virus X protein (HBx) induced hepatocellular carcinoma and its molecular mechanism. Methods By injecting HBx-enhanced green fluorescent protein (EGFP)-14-19 cells into the hepatic portal vein of Kunming mice, an animal model with long-term stable expression of HBx was constructed. Quantitative polymerase chain reaction (qPCR) was used to detect the expression of miRNA-544-3p in HBx-EGFP-14-19 cells and liver tissues of mouse models. miRNA-544-3p mimics and inhibitors were transiently transfected into HBx-EGFP-14-19 cells. The effects of miRNA-544-3p on the migration and invasion of HBx-EGFP-14-19 cells were detected by scratch wound healing assay and Transwell cell migration and invasion assay. qPCR and Western blotting were used to explore the regulatory effect of miRNA-544-3p on the expression of adenosine diphosphate ribosylation factor 6 (Arf6), protein kinase B (Akt) and mammalian target of rapamycin (mTOR). Results A mouse model that can stably express HBx for a long time was successfully constructed, and malignant tumors could be found in the liver tissues when the mice were sacrificed 360 d after modeling. Compared with the control group, the expression levels of miRNA-544-3p in HBx-EGFP-14-19 cells and HBx model mice at different time points (30, 90, 180, and 360 d after modeling) were decreased (all P<0.05). Compared with the control group, the migration and invasion abilities of HBx-EGFP-14-19 cells transfected with miRNA-544-3p mimics were decreased, and the migration and invasion abilities of HBx-EGFP-14-19 cells transfected with miRNA-544-3p inhibitors were increased (all P<0.05). The results of qPCR and Western blotting showed that the low expression of miRNA-544-3p was correlated with the up-regulation of Arf6 expression and the activation of Akt-mTOR signal axis. Conclusion HBx may promote the invasion and migration of mouse hepatocarcinoma cells by downregulating miRNA-544-3p and activating Arf6/Akt-mTOR signal axis.