Natural products extracted from plants are an alternative method for controlling postharvest citrus blue mold, caused by Penicillium italicum (P. italicum). In this study, RNA sequence analysis was used to investigate the underlying anti-fungal mechanism of flavonoids from Sedum aizoon L. (FSAL) on P. italicum. Significant differences in 3592 genes were observed, including 2507 up-regulated and 1085 down-regulated genes between the FSAL-treated and the control groups. Furthermore, the GO and KEGG analysis results indicated that FSAL inhibited genes related to the integral components of membrane, oxidation-reduction process, mitochondrion, ribosome, and amino acid metabolism. In the presence of FSAL, the cellular constituents, including DNA and RNA were leaked from hyphae of P. italicum. Reactive oxygen species (ROS) production in P. italicum was also determined with a significant concentration-effect under the treatment of FSAL. Thus, we speculate that the inhibitory activity of FSAL on P. italicum is mainly achieved by damaging the structure of the cell membrane and cell wall, disrupting the process of mitochondrial respiratory metabolism, protein biosynthesis, and amino acid metabolism, leading to cell death. The present study provided a global perspective on the molecular mechanism of FSAL on P. italicum through transcriptome analysis, which may help develop a novel plant-derived anti-fungal agent for the blue mold of citrus.