Previous report showed that cytosolic Ca²⁺ induced by hepatitis B virus X protein (HBx) promotes HBV replication. In this study, in vitro experiments showed that (i) HBV core assembly in vitro was promoted by Ca²⁺ through the sucrose density gradient and the analytical ultracentrifuge analysis. Also, (ii) transmission electron microscope analysis demonstrated these assembled HBV core particles were the capsids. Ex vivo experiments showed that the treatment of BAPTA-AM and cyclosporine A (CsA) reduced HBV capsids in the transfected HepG2 cells. In addition to that, the treatment of Thapsigargin (TG) increased HBV capsids in the transfected HepG2 cells. Furthermore, we investigated the increased HBV core assembly by HBx. The results show that the increased cytosolic calcium ions by HBx promote the HBV core assembly.