Ginsenoside Rg3, discovered from Red Panax ginseng, is a new anticancer agent, which shows inhibitive activities of tumor metastasis in mice and in vitro tumor cell invasion. There are two isomers for ginsenoside Rg3: 20-(R)-Rg3 and 20-(S)-Rg3. The research on the Rg3 structure is limitted, with no report on the use of Raman spectroscopy in such molecules. In the present article, the authors employed the Raman spectroscopy to study the structure of ginsenoside Rg3. Compared with 20-(R)-Rg3, the hydrocarbon chain of 20-(S)-Rg3 is located in the inner of molecules. In addition, there are significant differences in band location and relative intensity between the spectra of 20-(R)-Rg3 and 20-(S)-Rg3, and this indicates that Raman spectroscopy can be used as a simple, fast and accurate analytical tool to identify ginsenoside Rg3 isomers.