Si-wu decoction (SDE), a classic prescription in Traditional Chinese Medicine, has been used for the treatment of a variety of anaemia in China for centuries. In order to explore the scientific basis of the formula, we investigated the relationship between its chemical components and proliferation-promoting effects on rat marrow-derived mesenchymal stem cells (MSCs). Twenty (F-1-F-20) components were obtained and their proliferation-promoting effects on MSCs were investigated. The results showed that F-4, F-7, F-10, and F-11 stimulated the proliferation of the MSCs. The chemical components with proliferation-promoting effects on the MSCs were further identified by GC-MS, HPLC, LC-MS, and other spectra. Ligustilide (F-4) isolated from SDE showed the best proliferation-promoting effect. Palmitic acid methyl ester and stearic acid ethyl identified from F-7 and F-10 by HPLC were also confirmed to be responsible for stimulating MSC proliferation. A novel compound, 6,7-dihydroxy-3-(-prop-1-enyl)isobenzofuran-1(3H)-one, was found in the SDE for the first time by LC-MS(n), whose structure was similar to ligustilide.