Objective To investigate the effects of miR-583 on the replication and expression of hepatitis B virus (HBV) and related mechanisms. Methods Human liver cancer cells HepG2 and human liver cancer cells HepG2.2.15, which are stably transfected with HBV virus, were cultured in vitro. HepG2.2.15 and divided into blank control group, NC-siRNA group (transfected with NC-siRNA) and miR-583 siRNA group (transfected with miR-583 siRNA). Real time fluorescent quantitative PCR (qRT-PCR) method was used to detect the expression of miR-583, DDX5 mRNA and HBV DNA. The enzyme-linked immunoassay (ELISA) was used to detect the expression levels of hepatitis B virus antigen (HBsAg) and hepatitis B virus e antigen (HBeAg). The dual luciferase method was used to verify the targeting relationship between miR-583 and DDX5. Results Compared with HepG2 cells, the expression level of miR-583 in HepG2.2.15 cells was significantly increased (P＜0.05), and the expression level of DDX5 mRNA was significantly reduced(P＜0.05). Compared with the NC-siRNA group, the miR-583 expression level in HepG2.2.15 cells IN the miR-583 siRNA group was significantly reduced (P＜0.05), the DDX5 mRNA expression level was significantly increased (P＜0.05), and the expression levels of HBV DNA, HBsAg, and HBeAg in the cell supernatant were significantly reduced (P＜0.05). Target ScanHuman website prediction and dual luciferase experiment results showed that miR-583 and DDX5 had a targeted regulatory relationship. Conclusion MiR-583 can promote HBV replication and expression, which may be related to the targeted inhibition of DDX5 expression. Down regulating miR-583 expression can inhibit HBV replication and expression.