Inactivated mouse-brain-derived vaccines for Japanese encephalitis virus (JEV) have been used for many years. Recently, attempts have been made to employ cultured Vero cells to replace mouse brain tissues for developing cell-culture-derived vaccines that will be more suitable for worldwide usage. In this study, JEV replication processes in Vero and BHK cells and between stationary and microcarrier culture systems were investigated. Our results demonstrated that a stationary Vero cell culture system produced higher viral titers of JEV, including the Beijin-1 vaccine strain and the attenuated strain CH2195LA, than microcarrier culture did. BHK cells showed less significant differences in their replication kinetics between stationary and microcarrier cultures. Reducing serum concentration during infection led to an overall decrease of JEV production in Vero cells but an increase in BHK cells. By establishing a complete serum-free Vero cell culture, the microcarrier system resulted in a more than 4-log lowered yield compared to that of the stationary culture for JEV production. Thus, the stationary culture is the most efficient system for JEV production from cultured Vero cells.