Aims: This study examined the transformation pathways of ginsenosides G-Rb-1, G-Rb-3, and G-Rc by the fungus Fusarium sacchari. Methods and Results: Ginsenosides G-Rb-1, G-Rb-3 and G-Rc were isolated from leaves of Radix notoginseng, and their structural identification was confirmed using NMR. Transformation of G-Rb-1, G-Rb-3 and G-Rc by Fusarium sacchari was respectively experimented. Kinetic evolutions of G-Rb-1, G-Rb-3 and G-Rc and their metabolites during the cell incubation were monitored by HPLC analysis. High-performance liquid chromatography (HPLC) was used for monitoring the transformation kinetics of bioactive compounds during F. sacchari metabolism. Conclusions: Ginsenoside C-K was transformed by F. sacchari from G-Rb-1 via G-Rd or via G-F-2, or from G-Rb-1 via firstly Rd and then G-F-2, and C-Mx was transformed by F. sacchari or directly from Rb-3, or from Rb-3 via Gy-IX, while G-Mc was transformed by F. sacchari directly from G-Rc. Furthermore, C-K could be also formed from G-Rc via notoginsenoside Fe (N-Fe). Significance and Impact of the Study: The results showed an important practical application in the preparation of ginsenoside C-K. As our precious research indicated C-K possessed much more antitumor activities than C-Mx and G-Mc, so according to the transformation pathways proposed by this work, the production of antitumor compound C-K may be performed by biotransformation of G-Rb-1 previously isolated from PNLS.