Abstract The palindromic nonapeptide H‐ 1 RWQWRWQWR 9 ‐NH 2 (LfcinB (21–25) Pal ) was synthetize and scaled following the good manufacturing practices (GMP) guidelines to obtained batches up to 1 g of pure peptide which evidencing their synthetic viability. Its cytotoxic effect was tested against breast cancer cell lines MDA‐MB‐231 and MCF‐7 and was dependent on concentration against both. The IC 50 values were 135 μM and 66 μM, respectively. Against MCF‐7 the peptide exerts its greater cytotoxic effect at 135 μM, diminishing their cell viability to 21%. Furthermore, the palindromic peptide did not exhibit a significant cytotoxic effect against the nontumorigenic cells MCF‐12, BMEC (bovine mammary epithelial cells) or fibroblast, which confirms its selectivity. The cancerous cells treated at 135 μM for 2 h exhibited morphological changes like cellular shrinkage and rounded forms. Trough flow cytometry assays we evidence that 73% of the events were related to late apoptosis, and cells that exhibited mitochondrial membrane depolarization also had an increase in the relative expression of caspase‐3. Our results suggest that the LfcinB (21–25) Pal peptide has a cytotoxic effect against MCF‐7 mainly through apoptotic events which makes it a promising candidate for therapeutic agent.