To examine the effect of calcium and magnesium on hepatitis E virus (HEV) infection in PLC/PRF/5 cells, quantitative real-time fluorescent RT-PCR and ELISA were used to detect the viral nucleic acid and antigen content of HEV. The virus nucleic acid and ORF2 antigen in the cells and supernatant were examined at different time points after HEV infection in the presence of EGTA, calcium or magnesium. The addition of calcium and magnesium to the cell culture medium increased virus binding, whereas chelating agents inhibited HEV binding 1 to 24 h after HEV incubation. Both calcium and magnesium increased the production of HEV in the cell culture medium of PLC/PRF/5 cells after two to five weeks, while calcium had more obvious effect than magnesium. Calcium and magnesium could promote HEV infection in PLC/PRF/5 cells. The addition of calcium and magnesium in the HEV culture is helpful for the production of viruses.