The anticancer potential of some antimicrobial peptides has been reported. Hs02 is a recently characterized Intragenic Antimicrobial Peptide (IAP), which was able to exhibit potent antimicrobial and anti-inflammatory action. In this study, we evaluate for the first time the antineoplastic potential of IAP Hs02 using cell lines representing the main types of leukemia as cancer models. Interestingly, this peptide decreased the viability of several leukemic cell lines, without compromising the viability of PBMCs. In the HL-60 line, treatment with Hs02 controlled cell division, leading to cell arrest in the G1 phase of the cell cycle. More importantly, HL-60 cells treated with Hs02 undergo cell death, with the formation of pores in the plasma membrane and the release of LDH. Accordingly, Hs02 treatment stimulated the expression of components involved in pyroptosis, such as NLRP1, CASP-1, GSDME, and IL-1β. Taken together, our data characterize the antineoplastic potential of Hs02 and open na opportunity for both evaluating the peptide's antineoplastic potential in other cancer models and using this molecule as a template for new peptides with therapeutic potential against cancer.