Enterocytozoon hepatopenaei (EHP) is an emerging pathogen threatening the prawn aquaculture industry globally, due to losses resulting from retarded growth. Since the first detection of EHP in Thailand, EHP has been reported from other countries in Asia and South America. However, the mechanism of EHP spread into new countries and/or areas has not been established. It is uncertain whether infectious EHP can be inadvertently introduced via frozen commodity prawns. Therefore, this study investigated the effect of storage of prawns at commercial freezing temperatures (at or below -18 °C) has on EHP infectivity. We assessed the ability of EHP to cause infection following oral challenge of Penaeus vannamei with EHP-infected hepatopancreas that had been frozen at -18 °C for 0 h, 24 h, 7 days, or 14 days. Our results showed that freezing at -18 °C for 7 days and 14 days further reduces EHP in prawn tissue (hepatopancreas) and inhibits transmission. These findings suggest that freezing prawns at -18 °C for at least 7 days might prevent spread of EHP into new countries via commodity prawns.