Jaspamide, a natural peptide isolated from the marine sponge Hemiastrella minor, was used in the study of polyploidy in HL-60 cells. Jaspamide at 5 × 10–8 M concentration exhibited antiproliferative activity and an increased CD4 and CD14 surface expression. After 2 days of cultivation, 56.3% of the exposed cells became multinuclear compared with 2.4% in controls. Moreover, the size and the number of nuclei of the cells increased in a time-dependent manner. An increased number of metaphase chromosomes was observed by microscopical enumeration after colcemid treatment for 60 min. The analysis of the DNA content of these cells, measured by propidium iodide staining, revealed a significant increase in the cells percentage with increased DNA content. Taken together, these findings indicate that the jaspamide treatment induces polyploidization in the HL-60 cell line.