Following the three-stage approach of the WOAH (World Organisation for Animal Health) standard on the confirmation of susceptible species to a specific pathogen, Artemia franciscana juveniles were challenged with immersion exposure (IM) in a homogenate suspension of Penaeus vannamei hepatopancreas infected with Ecytonucleospora hepatopenaei (EHP) and per os administration (PO) with chlorella soaked in EHP-infected hepatopancreatic homogenate suspension. Using TaqMan probe-based quantitative PCR, we detected that loads of EHP in A. franciscana juveniles from both the IM and PO groups were about 101 copies/ng-DNA on the 3rd day post-exposure (3 dpe). However, no more EHP was detectable in the challenged A. franciscana juveniles on 7 dpe. Nested PCR targeting the EHP spore wall protein gene did not produce a target band in A. franciscana juveniles on either 3 dpe or 7 dpe. The confirmative evidence of the absence of sustained infection with EHP in the exposed Artemia was substantiated using in situ DIG-labeling-loop-mediated DNA amplification (ISDL) on A. franciscana juvenile sections, which revealed a few weak grey-blue positive signals appeared on 3 dpe but disappeared on 7 dpe. The results indicated that exposure to EHP resulted in the invasion of Artemia in intestinal epithelial cells, but the infection was unable to persist, confirming Artemia is not susceptible to EHP. Therefore, the research countered the assertion of Artemia as a potential EHP reservoir.