The role of the anti-cancer agent Viscum album agglutinin-I (VAA-I) in leukaemia PLB-985 cells differentiated toward a neutrophil-like phenotype by dimethylsulphoxide (PLB-985D) has never been studied. This study investigated whether or not VAA-I can induce cytoskeletal breakdown in PLB-985D cells, as previously observed in undifferentiated PLB-985 cells. VAA-I was found to induce apoptosis in PLB-985D cells, as assessed by cytology and by degradation of gelsolin, an event known to occur via caspase-3 activation. VAA-I induced cytoskeletal breakdown based on the disruption of the F-actin network and cleavage of paxillin, vimentin and lamin B(1). In addition, we demonstrated, for the first time, that non-muscle myosin heavy chain IIA (NMHC-IIA) was cleaved by VAA-I treatment. Degradation of NMHC-IIA was reversed by the pan caspase inhibitor z-VAD-fmk in PLB-985D cells and neutrophils. However, unlike lamin B(1), no NMHC-IIA was detected on the cell surface of apoptotic neutrophils. In conclusion, PLB-985D cells responded in a similar manner to neutrophils regarding the degradation of the tested cytoskeletal. Therefore, PLB-985D cells may provide a suitable substitute for neutrophils in screening experiments, preventing extensive neutrophil cell isolation.