A transcriptomic analysis was conducted to elucidate the mechanism of action of the antimicrobial peptide D-R4F4 against Escherichia coli. Cultures were treated with the peptide at its MIC for 24 h, with PBS controls included. Total RNA was extracted, quality-checked, and rRNA-depleted to enrich mRNA. Strand-specific cDNA libraries were prepared through fragmentation, adapter ligation, and PCR amplification, followed by purification and quality assessment. Sequencing was performed on the Illumina HiSeq platform (Novogene, UK). The resulting dataset provides a genome-wide view of differential gene expression, offering key insights into the molecular pathways affected by D-R4F4 exposure.