Viral hemorrhagic septicemia virus (VHSV) is an aquatic rhabdovirus causing severe disease in numerous freshwater and saltwater fish species. Initially the virus was been found to cause disease in European fish populations starting around 1938 and was first detected in North America in the late 1980s. Of the four VHSV genotypes (I, II, III, and IV), the North American subtype IVb isolates have a broad host range. To determine whether endangered pallid sturgeon Scaphirhynchus albus, are susceptible to VHSV-IVb infection, juvenile pallid sturgeon and two pallid sturgeon cell lines derived from skin and spleen tissue were tested. Detection of viable virus via a plaque assay and molecular detection of the virus results by a RT-PCR (reverse transcription-polymerase chain reaction) confirmed VHSV-IVb in vitro replication in pallid sturgeon cell lines. Pallid sturgeon were also susceptible to VHSV-IVb infection when exposed to the virus by immersion at concentration of 5 X 10e5 plaque forming units per milliliter (pfu/ml) and by injection at a dose of 1 X 10e6 plaque forming units per fish (pfu/fish) during 28-day long challenge experiments. However, after widespread mortality occurred in all treatment groups, including control fish, it was determined that the pallid sturgeon stock fish were infected with Missouri River Sturgeon Iridovirus (MRSIV) prior to experimental challenge. Nevertheless, VHSV-exposed fish suffered equal or higher mortalities (38 – 48%) than mock treated (MRSIV-infected) fish (29 – 38%) and histopathology samples showed reduced hematopoietic cells in spleen and kidney tissues and hemorrhage in the gastrointestinal organs only in VHSV-treated fish. These results suggest that pallid sturgeon are susceptible to VHSV-IVb infection, but the degree of pathogenicity was confounded by the underlying MRSIV infection. Our data also suggest that pallid sturgeon may serve as carriers of VHSV because the virus was isolated from surviving fish that showed no clinical signs, yet were positive for both VHSV and MRSIV. Research comparing susceptibility of pathogen-free and MRSIV-infected fish to VHSV-IVb is needed to accurately assess the vulnerability of pallid sturgeon to VHSV-IVb.