Penicillium expansum (P. expansum) is a harmful pathogenic mold responsible for huge postharvest losses in apples. Epsilon‐poly‐L‐lysine (ε-PL) is a non-toxic antiseptic peptide derived from Streptomyces albulus. Many food preservative strategies use ε-PL to suppress foodborne pathogens in condiments, cakes, fruit and vegetable juices. In the present research, ε-PL’s inhibitory effects against P. expansum in vitro and in apples were investigated. The molecular mechanisms of ε-PL inhibiting P. expansum in apples were investigated by transcriptome analysis. Results showed that doses of 600 mg L−1 of ε-PL could prevent the development of P. expansum in vitro and control blue mold decay in apples. Plausible mechanisms of ε-PL against P. expansum in apples include: (i) reduction of antioxidant potential, including decreasing the expression of genes encoding superoxide dismutase (SOD), catalase (CAT), glutathione s-transferase (GST), xanthine dehydrogenase (XDH), D-amino-acid oxidase (DAO), weakening ROS scavenging ability of P. expansum cells and causing oxidative damage to cell, (ii) weakening its virulence towards apples, including reducing the expression of genes coding for cell wall degrading enzymes (CWDEs) such as pectinase and cellulase, (iii) disrupting mitochondrial function of P. expansum, including reducing expression of several rate-limiting enzyme coding genes of energy metabolic pathways such as EMP, TCA cycle and oxidative phosphorylation, which interfered with the cells’ energy metabolism, leading to insufficient ATP synthesis, weakening P. expansum’s resistance to external stress, and finally slowing down the invasion process into apples, (ⅳ) damaging the membrane integrity of P. expansum, including reducing the expression of genes encoding diaphragm wall degradation enzymes, cytomembrane lipid and ergosterol synthetase, multi-drug transporter, and inhibiting the differentiation of conidia.