Penicillium digitatum is the most damaging pathogen provoking green mold in citrus fruit during storage, and there is an urgent need for novel antifungal agents with high efficiency. The aim of this study was to investigate the antifungal effects of peptide thanatin against P. digitatum and the molecular mechanisms. Results showed that peptide thanatin had a prominent inhibitory effect on P. digitatum by in vitro and in vivo test. A total of 938 genes, including 556 downregulated and 382 upregulated genes, were differentially expressed, as revealed by RNA-seq of whole P. digitatum genomes analysis with or without thanatin treatment. The downregulated genes mainly encoded RNA polymerase, ribosome biogenesis, amino acid metabolism, and major facilitator superfamily. The genes associated with heat shock proteins and antioxidative systems were widely expressed in thanatin-treated group. DNA, RNA, and the protein content of P. digitatum were significantly decreased after thanatin treatment. In conclusion, thanatin could inhibit the growth of P. digitatum, and the underlying mechanism might be the genetic information processing and stress response were affected. The research will provide more precise and directional clues to explore the inhibitory mechanism of thanatin on growth of P. digitatum.