Haemophilus parasuis is the causative agent of Glasser’s disease in pigs. Cytolethal distending toxin (CDT) is an important virulence factor of H. parasuis. It is composed of three subunits: CdtA, CdtB and CdtC and all were successfully expressed in soluble form in Escherichia coli when the signal peptides were removed. Purified CdtB had DNase activity, i.e. caused DNA double strand damage, in vitro and in vivo prior to cell arrest and apoptosis. Flow cytometry analysis showed CdtB alone could induce cell cycle arrest and apoptosis in PK-15 porcine kidney and pulmonary alveolar macrophage (PAM) cells, which could be enhanced by CdtA or/and CdtC. CDT holotoxin could lead to significant cell distension, G2 arrest and apoptotic death in PK-15 and PAM cells. The apoptosis induced by CDT holotoxin was significantly inhibited by pifithrin-α, which indicates that it is p53-dependent. The results suggest that H. parasuis CDT holotoxin is a major virulence factor.