Abstract: A total of 150 purified isolates of Pasteurella multocida serotype B which has been used in previous (Salmah, 2004) were further carried out for plasmid DNA curing experiment to determine hyaluronidase activity, antibiotic resistance pattern (ARP) and mice lethality test (LD50) for their role of pathogenicity. A plasmid curing experiment was carried out by using the intercalating agent; ethidium bromide and rifampicin, where it was found all the plasmids had been ‘cured` (plasmidless) from Pasteurella multocida. All of these plasmidless isolates remained their phenotypic characteristics. They shown the same antibiotic resistance pattern as before curing, produced hyaluronidase and possessed lethality activity in mice when injected intraperitoneally (i.p). Based on this observation, the antibiotic resistance, hyaluronidase activity and mice virulence could probably chromosomal-mediated. This plasmid might not played any role in pathogenicity of Pasteurella multocida serotype B, however this information is important for the construction of shuttle vectors in genetic studies of the pathogenicity of haemorrhagic septicaemia (HS).