Aims: The effects of medium composition, calcium, iron and oxygen tension on the haemolytic activity of Plesiomonas shigelloides were investigated. Methods and Results: The haemolytic activity of seven strains of Ple. shigelloides was tested on the surface of Luria Agar (LA), Brain Heart Infusion Agar (BHIA) and Trypitic Soy Agar (TSA) containing 5% (v/v) sheep blood, and in the Agar Overlay (AO) assay. All strains produced beta -haemolysis in the AO assay in three media, and on the surface of LA. The kinetics of growth and haemolytic activity of Ple. shigelloides 9P3-1 were evaluated in six different media, and the highest production of haemolysin occurred in Luria Broth (LB). The haemolytic activity of 9P3-1 was stimulated by Ca2+ and inhibited by EDTA. Addition of iron to the culture medium did not affect bacterial growth, although it reduced bacterial haemolytic activity. In the presence of an iron chelator, growth of the 9P3-1 was inhibited, but its haemolytic activity was enhanced. Conclusions: The haemolytic activity of Ple. shigelloides depends on medium composition, and that it is regulated by iron and is calcium-dependent. Significance and Impact of the Study: These results show the importance of optimization of media composition and oxygen tension for detection of Ple. shigelloides haemolytic activity.