Protopanaxadiol (PPD)-type ginsenosides are the main ginsenosides in ginseng (Panax ginseng C. A. Meyer) with potential therapeutic effects on diseases related to intestinal flora imbalance. This study aimed to investigate the in vitro metabolism of protopanaxadiol ginsenosides in human intestinal flora and their effect on the flora. Rapid resolution liquid chromatography coupled with quadruple-time-of-flight mass spectrometry (RRLC-Q-TOF MS) was utilised for the transformation of ginsenoside constituents for sample identification. Using 16S rDNA gene sequencing technique, the effect of PPD-type ginsenosides on gut microflora was analysed based on the indices of microflora diversity and gut microflora. The sample was transformed for 6 h, and the metabolites were ginsenoside Rb1, Rc, Rb2, Rb3, CO, Gyp-IX, Gyp-XVII, CMc-1, F2, Rg3, CK, Rh2, and PPD. The metabolites were CK, Rh2, and PPD when the samples were transformed for 60 h. The intestinal microflora were subjected to high-throughput sequencing using the Illumina MiSeq 2500 sequencing platform. In comparison with the faecal sample from the blank group, the protopanaxadiol saponin group significantly increased the relative abundance of Firmicutes and significantly decreased Bacteroidetes and Proteobacteria at the phylum level, whereas it significantly increased the relative abundance of Prevotella_9, Faecalibacterium, and Dialister and significantly decreased Escherichia-Shigella, Dorea, and Lachnoclostridium at the genus level. This study provides a basis for the determination of the pharmacodynamic material basis and pharmacodynamic targets of PPD-type ginsenosides based on the intestinal flora.